Porz, an essential component of the type ix secretion system of porphyromonas gingivalis, delivers anionic lipopolysaccharide to the poru sortase for transpeptidase processing of t9ss cargo proteins

Mariusz Madej; Zuzanna Nowakowska; Miroslaw Ksiazek; Anna M. Lasica; Danuta Mizgalska; Magdalena Nowak; Anna Jacula; Monika Bzowska; Carsten Scavenius; Jan J. Enghild; Joseph Aduse-Opoku; Michael A. Curtis; F. Xavier Gomis-Rüth; Jan Potempa

doi:10.1128/mBio.02262-20

Porz, an essential component of the type ix secretion system of porphyromonas gingivalis, delivers anionic lipopolysaccharide to the poru sortase for transpeptidase processing of t9ss cargo proteins

Mariusz Madej, Zuzanna Nowakowska, Miroslaw Ksiazek, Anna M. Lasica, Danuta Mizgalska, Magdalena Nowak, Anna Jacula, Monika Bzowska, Carsten Scavenius, Jan J. Enghild, Joseph Aduse-Opoku, Michael A. Curtis, F. Xavier Gomis-Rüth^*, Jan Potempa

^*Corresponding author for this work

Centre for Host Microbiome Interactions

Research output: Contribution to journal › Article › peer-review

17 Citations (Scopus)

Abstract

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggest-ing that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttransla-tional modification and retention of T9SS cargos on the bacterial surface. IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis. Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.

Original language	English
Article number	e02262-20
Pages (from-to)	1-14
Number of pages	14
Journal	Mbio
Volume	12
Issue number	1
DOIs	https://doi.org/10.1128/mBio.02262-20
Publication status	Published - 1 Jan 2021

Keywords

Gingipains
Lipopolysaccharide
Porphyromonas gingivalis
Secretion
T9SS

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1128/mBio.02262-20

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Madej, M., Nowakowska, Z., Ksiazek, M., Lasica, A. M., Mizgalska, D., Nowak, M., Jacula, A., Bzowska, M., Scavenius, C., Enghild, J. J., Aduse-Opoku, J., Curtis, M. A., Xavier Gomis-Rüth, F., & Potempa, J. (2021). Porz, an essential component of the type ix secretion system of porphyromonas gingivalis, delivers anionic lipopolysaccharide to the poru sortase for transpeptidase processing of t9ss cargo proteins. Mbio, 12(1), 1-14. Article e02262-20. https://doi.org/10.1128/mBio.02262-20

@article{3ec11b44daf2471fade119dd3096e463,

title = "Porz, an essential component of the type ix secretion system of porphyromonas gingivalis, delivers anionic lipopolysaccharide to the poru sortase for transpeptidase processing of t9ss cargo proteins",

abstract = "Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggest-ing that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttransla-tional modification and retention of T9SS cargos on the bacterial surface. IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis. Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.",

keywords = "Gingipains, Lipopolysaccharide, Porphyromonas gingivalis, Secretion, T9SS",

author = "Mariusz Madej and Zuzanna Nowakowska and Miroslaw Ksiazek and Lasica, {Anna M.} and Danuta Mizgalska and Magdalena Nowak and Anna Jacula and Monika Bzowska and Carsten Scavenius and Enghild, {Jan J.} and Joseph Aduse-Opoku and Curtis, {Michael A.} and {Xavier Gomis-R{\"u}th}, F. and Jan Potempa",

note = "Funding Information: This study was supported in part by grants from Spanish and Catalan public and private bodies (PID2019-107725RG-I00, 2017SGR3, and Fundaci{\'o} “La Marat{\'o} de TV3” 201815 to F.X.G.-R.); National Science Centre, Poland grants UMO-2015/19/N/NZ1/ 00322 to M.M., UMO-2012/04/A/NZ1/00051 to J.P., and UMO-2016/23/N/NZ1/01513 to Z.N.; Polish Ministry of Science and Higher Education grant 1306/MOB/IV/2015/0 to M. K.; U.S. National Institutes of Health, NIDCR, grants DE09761 and DE026280 to J.P.; and Medical Research Council grant MR/P012175/2 to M.A.C. Publisher Copyright: {\textcopyright} 2021 Madej et al. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

year = "2021",

month = jan,

day = "1",

doi = "10.1128/mBio.02262-20",

language = "English",

volume = "12",

pages = "1--14",

journal = "Mbio",

issn = "2161-2129",

publisher = "American Society for Microbiology",

number = "1",

}

Madej, M, Nowakowska, Z, Ksiazek, M, Lasica, AM, Mizgalska, D, Nowak, M, Jacula, A, Bzowska, M, Scavenius, C, Enghild, JJ, Aduse-Opoku, J , Curtis, MA, Xavier Gomis-Rüth, F & Potempa, J 2021, 'Porz, an essential component of the type ix secretion system of porphyromonas gingivalis, delivers anionic lipopolysaccharide to the poru sortase for transpeptidase processing of t9ss cargo proteins', Mbio, vol. 12, no. 1, e02262-20, pp. 1-14. https://doi.org/10.1128/mBio.02262-20

Porz, an essential component of the type ix secretion system of porphyromonas gingivalis, delivers anionic lipopolysaccharide to the poru sortase for transpeptidase processing of t9ss cargo proteins. / Madej, Mariusz; Nowakowska, Zuzanna; Ksiazek, Miroslaw et al.
In: Mbio, Vol. 12, No. 1, e02262-20, 01.01.2021, p. 1-14.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Porz, an essential component of the type ix secretion system of porphyromonas gingivalis, delivers anionic lipopolysaccharide to the poru sortase for transpeptidase processing of t9ss cargo proteins

AU - Madej, Mariusz

AU - Nowakowska, Zuzanna

AU - Ksiazek, Miroslaw

AU - Lasica, Anna M.

AU - Mizgalska, Danuta

AU - Nowak, Magdalena

AU - Jacula, Anna

AU - Bzowska, Monika

AU - Scavenius, Carsten

AU - Enghild, Jan J.

AU - Aduse-Opoku, Joseph

AU - Curtis, Michael A.

AU - Xavier Gomis-Rüth, F.

AU - Potempa, Jan

N1 - Funding Information: This study was supported in part by grants from Spanish and Catalan public and private bodies (PID2019-107725RG-I00, 2017SGR3, and Fundació “La Marató de TV3” 201815 to F.X.G.-R.); National Science Centre, Poland grants UMO-2015/19/N/NZ1/ 00322 to M.M., UMO-2012/04/A/NZ1/00051 to J.P., and UMO-2016/23/N/NZ1/01513 to Z.N.; Polish Ministry of Science and Higher Education grant 1306/MOB/IV/2015/0 to M. K.; U.S. National Institutes of Health, NIDCR, grants DE09761 and DE026280 to J.P.; and Medical Research Council grant MR/P012175/2 to M.A.C. Publisher Copyright: © 2021 Madej et al. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/1/1

Y1 - 2021/1/1

N2 - Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggest-ing that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttransla-tional modification and retention of T9SS cargos on the bacterial surface. IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis. Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.

AB - Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggest-ing that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttransla-tional modification and retention of T9SS cargos on the bacterial surface. IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis. Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.

KW - Gingipains

KW - Lipopolysaccharide

KW - Porphyromonas gingivalis

KW - Secretion

KW - T9SS

UR - http://www.scopus.com/inward/record.url?scp=85101308058&partnerID=8YFLogxK

U2 - 10.1128/mBio.02262-20

DO - 10.1128/mBio.02262-20

M3 - Article

AN - SCOPUS:85101308058

SN - 2161-2129

VL - 12

SP - 1

EP - 14

JO - Mbio

JF - Mbio

IS - 1

M1 - e02262-20

ER -

Porz, an essential component of the type ix secretion system of porphyromonas gingivalis, delivers anionic lipopolysaccharide to the poru sortase for transpeptidase processing of t9ss cargo proteins

Abstract

Keywords

UN SDGs

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