Structural and functional analyses of the Porphyromonas gingivalis type IX secretion system PorN protein

Abstract

Porphyromonas gingivalis, the major human pathogen bacterium associated with periodontal diseases, secretes virulence factors through the Bacteroidetes-specific type IX secretion system (T9SS). Effector proteins of the T9SS are recognized by the complex via their conserved C-terminal domains (CTDs). Among the 18 proteins essential for T9SS function in P. gingivalis, PorN is a periplasmic protein that forms large ring-shaped structures in association with the PorK outer membrane lipoprotein. PorN also mediates contacts with the PorM subunit of the PorLM energetic module, and with the effector’s CTD. However, no information is available on the PorN structure and on the implication of PorN domains for T9SS assembly and effector recognition. Here we present the crystal structure of PorN at 2.0-Å resolution, which represents a novel fold with no significant similarity to any known structure. In agreement with in silico analyses, we also found that the N- and C-terminal regions of PorN are intrinsically disordered. Our functional studies showed that the N-terminal disordered region is involved in PorN dimerization while the C-terminal disordered region is involved in the interaction with PorK. Finally, we determined that the folded PorN central domain is involved in the interaction with PorM, as well as with the effector’s CTD. Altogether, these results lay the foundations for a more comprehensive model of T9SS architecture and effector transport. Porphyromonas gingivalis, the major human pathogen bacterium associated with periodontal diseases, secretes virulence factors through the Bacteroidetes-specific type IX secretion system (T9SS). Effector proteins of the T9SS are recognized by the complex via their conserved C-terminal domains (CTDs). Among the 18 proteins essential for T9SS function in P. gingivalis, PorN is a periplasmic protein that forms large ring-shaped structures in association with the PorK outer membrane lipoprotein. PorN also mediates contacts with the PorM subunit of the PorLM energetic module, and with the effector’s CTD. However, no information is available on the PorN structure and on the implication of PorN domains for T9SS assembly and effector recognition. Here we present the crystal structure of PorN at 2.0-Å resolution, which represents a novel fold with no significant similarity to any known structure. In agreement with in silico analyses, we also found that the N- and C-terminal regions of PorN are intrinsically disordered. Our functional studies showed that the N-terminal disordered region is involved in PorN dimerization while the C-terminal disordered region is involved in the interaction with PorK. Finally, we determined that the folded PorN central domain is involved in the interaction with PorM, as well as with the effector’s CTD. Altogether, these results lay the foundations for a more comprehensive model of T9SS architecture and effector transport. Porphyromonas gingivalis, an anaerobic, nonmotile gram-negative bacterium, is the major human oral pathogen associated with periodontal diseases (1Bostanci N. Belibasakis G.N. Porphyromonas gingivalis: An invasive and evasive opportunistic oral pathogen.FEMS Microbiol. Lett. 2012; 333: 1-9Google Scholar, 2Lunar Silva I. Cascales E. Molecular strategies underlying Porphyromonas gingivalis virulence.J. Mol. Biol. 2021; 433: 166836Google Scholar). Chronic P. gingivalis infection is also linked to rheumatoid arthritis, heart disease, diabetes, Alzheimer disease, and other systemic diseases (3Garcia R.I. Henshaw M.M. Krall E.A. Relationship between periodontal disease and systemic health.Periodontol. 2000. 2001; 25: 21-36Google Scholar, 4Bahekar A.A. Singh S. Saha S. Molnar J. Arora R. The prevalence and incidence of coronary heart disease is significantly increased in periodontitis: A meta-analysis.Am. Heart J. 2007; 154: 830-837Google Scholar, 5Meyer M.S. Joshipura K. Giovannucci E. Michaud D.S. A review of the relationship between tooth loss, periodontal disease, and cancer.Cancer Causes Control. 2008; 19: 895-907Google Scholar, 6Singhrao S.K. Harding A. Poole S. Kesavalu L. Crean S. Porphyromonas gingivalis periodontal infection and its putative links with Alzheimer’s disease.Mediators Inflamm. 2015; 2015: 1-10Google Scholar, 7Olsen I. Singhrao S.K. Potempa J. Citrullination as a plausible link to periodontitis, rheumatoid arthritis, atherosclerosis and Alzheimer’s disease.J. Oral Microbiol. 2018; 10: 1487742Google Scholar, 8Eriksson K. Fei G. Lundmark A. Benchimol D. Lee L. Hu Y.O.O. Kats A. Saevarsdottir S. Catrina A.I. Klinge B. Andersson A.F. Klareskog L. Lundberg K. Jansson L. Yucel-Lindberg T. Periodontal health and oral microbiota in patients with rheumatoid arthritis.J. Clin. Med. 2019; 8: 630Google Scholar, 9Perricone C. Ceccarelli F. Matteo S. Di Carlo G. Bogdanos D.P. Lucchetti R. Pilloni A. Valesini G. Polimeni A. Conti F. Porphyromonas gingivalis and rheumatoid arthritis.Curr. Opin. Rheumatol. 2019; 31: 517-524Google Scholar). Virulence factors responsible for P. gingivalis pathogenesis are secreted through the Bacteroidetes-specific type IX secretion system (T9SS) (10McBride M.J. Zhu Y. Gliding motility and Por secretion system genes are widespread among members of the phylum Bacteroidetes.J. Bacteriol. 2013; 195: 270-278Google Scholar, 11McBride M.J. Bacteroidetes gliding motility and the type IX secretion system.Microbiol. Spectr. 2019; 7https://doi.org/10.1128/microbiolspec.PSIB-0002-2018Google Scholar, 12Gorasia D.G. Veith P.D. Reynolds E.C. The type IX secretion system: Advances in structure, function and organisation.Microorganisms. 2020; 8: 1173Google Scholar). These virulence factors are mainly cysteine proteases called gingipains that degrade host cell tissues and interfere with innate host defense mechanisms (13Hajishengallis G. Periodontitis: From microbial immune subversion to systemic inflammation.Nat. Rev. Immunol. 2015; 15: 30-44Google Scholar). Among other virulence factors secreted by the T9SS are the heme-binding protein Hbp35 involved in extracellular iron uptake (14Shoji M. Sato K. Yukitake H. Kondo Y. Narita Y. Kadowaki T. Naito M. Nakayama K. Por secretion system-dependent secretion and glycosylation of Porphyromonas gingivalis hemin-binding protein 35.PLoS One. 2011; 6e21372Google Scholar) and the peptidylarginine deiminase PPAD whose citrullination activity is linked to rheumatoid arthritis (15Montgomery A.B. Kopec J. Shrestha L. Thezenas M.-L. Burgess-Brown N.A. Fischer R. Yue W.W. Venables P.J. Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: Implications for autoimmunity in rheumatoid arthritis.Ann. Rheum. Dis. 2016; 75: 1255-1261Google Scholar). T9SS acts as a two-step mechanism: the substrate proteins are first exported into the periplasm by the Sec machinery after cleavage of their N-terminal signal peptide; then, a conserved C-terminal domain (CTD) is specifically recognized by the T9SS that transports them across the outer membrane (OM) (16Seers C.A. Slakeski N. Veith P.D. Nikolof T. Chen Y.-Y. Dashper S.G. Reynolds E.C. The RgpB C-terminal domain has a role in attachment of RgpB to the outer membrane and belongs to a novel C-terminal-domain family found in Porphyromonas gingivalis.J. Bacteriol. 2006; 188: 6376-6386Google Scholar, 17Veith P.D. Nor Muhammad N.A. Dashper S.G. Likić V.A. Gorasia D.G. Chen D. Byrne S.J. Catmull D.V. Reynolds E.C. Protein substrates of a novel secretion system are numerous in the Bacteroidetes phylum and have in common a cleavable C-terminal secretion signal, extensive post-translational modification, and cell-surface attachment.J. Proteome Res. 2013; 12: 4449-4461Google Scholar). Once outside the cell, the substrates are either delivered into the extracellular medium or remain attached to the cell surface through anchoring to anionic lipopolysaccharide (18Glew M.D. Veith P.D. Peng B. Chen Y.-Y. Gorasia D.G. Yang Q. Slakeski N. Chen D. Moore C. Crawford S. Reynolds E.C. PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis.J. Biol. Chem. 2012; 287: 24605-24617Google Scholar, 19Gorasia D.G. Veith P.D. Chen D. Seers C.A. Mitchell H.A. Chen Y.-Y. Glew M.D. Dashper S.G. Reynolds E.C. Porphyromonas gingivalis type IX secretion substrates are cleaved and modified by a sortase-like mechanism.PLoS Pathog. 2015; 11e1005152Google Scholar, 20Veith P.D. Glew M.D. Gorasia D.G. Reynolds E.C. Type IX secretion: The generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers.Mol. Microbiol. 2017; 106: 35-53Google Scholar, 21Madej 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. Gomis-Rüth F.X. Potempa J. 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. 2021; 12e02262-20Google Scholar). Beside P. gingivalis, the T9SS is also encoded on the genome of a large subset of Bacteroidetes species, including the model bacterium Flavobacterium johnsoniae (10McBride M.J. Zhu Y. Gliding motility and Por secretion system genes are widespread among members of the phylum Bacteroidetes.J. Bacteriol. 2013; 195: 270-278Google Scholar). In this nonpathogenic soil bacterium, the T9SS contributes to gliding motility by secreting SprB, a cell surface adhesin organized as filaments and that is required for movement on solid surfaces (22Nelson S.S. Bollampalli S. McBride M.J. SprB is a cell surface component of the Flavobacterium johnsoniae gliding motility machinery.J. Bacteriol. 2008; 190: 2851-2857Google Scholar). Studies on F. johnsoniae motility led to the model that the T9SS is a new rotary machine driven by proton motive force powering SprB rotation (23Shrivastava A. Lele P.P. Berg H.C. A rotary motor drives Flavobacterium gliding.Curr. Biol. 2015; 25: 338-341Google Scholar). To date, 18 genes have been identified as essential for T9SS function in P. gingivalis (12Gorasia D.G. Veith P.D. Reynolds E.C. The type IX secretion system: Advances in structure, function and organisation.Microorganisms. 2020; 8: 1173Google Scholar, 24Lasica A.M. Ksiazek M. Madej M. Potempa J. The type IX secretion system (T9SS): Highlights and recent insights into its structure and function.Front. Cell. Infect. Microbiol. 2017; 7: 215Google Scholar). Among them, the porK-L-M-N genes (gldK-L-M-N in F. johnsoniae) are cotranscribed (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar). The encoded by these genes into a complex the an complex through which are across the and of the the and that is to the to the attachment responsible for effector processing and attachment at the cell surface or in the medium (12Gorasia D.G. Veith P.D. Reynolds E.C. The type IX secretion system: Advances in structure, function and organisation.Microorganisms. 2020; 8: 1173Google Scholar, 20Veith P.D. Glew M.D. Gorasia D.G. Reynolds E.C. Type IX secretion: The generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers.Mol. Microbiol. 2017; 106: 35-53Google Scholar). The as a complex that to the T9SS complex K. Naito M. Yukitake H. H. M. McBride M.J. Nakayama K. A protein secretion system linked to gliding motility and S. A. Scholar). complex is to the to secreted via contacts between the effector and the PorM and PorN proteins M.S. M. Cascales E. A conserved of Porphyromonas type IX secretion C-terminal secretion signal with the 2018; Scholar). complex in the membrane complex and the and PorM are membrane proteins that through their (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar). has a PorM an periplasmic domain that of the periplasmic P. J. M.S. A. Cascales E. Kellenberger C. Cambillau C. Roussel A. Type IX secretion system PorM and gliding machinery the periplasmic 2018; Scholar, K. K. Nakayama K. K. PorM, a component of bacterial type IX secretion forms a with a Res. 2020; Scholar). In F. the complex to the rotary motor associated with gliding motility R. A. F. A. F. S. and of the motor that type secretion and gliding Microbiol. 2021; Scholar). a structure of this complex by a and and functional with known R. A. F. A. F. S. and of the motor that type secretion and gliding Microbiol. 2021; Scholar). The complex with the through contacts between the PorM periplasmic domain and PorN and PorK (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar, P. J. M.S. A. Cascales E. Kellenberger C. Cambillau C. Roussel A. Type IX secretion system PorM and gliding machinery the periplasmic 2018; Scholar). PorN is a periplasmic PorK is an (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar). and that the complex into large ring-shaped structures of D.G. Veith P.D. Glew M.D. Sato K. Yukitake H. Nakayama K. Reynolds E.C. insights into the PorK and PorN of the Porphyromonas gingivalis type IX secretion Pathog. 2016; Scholar, D.G. G. Seers C.A. C.A. Glew M.D. McBride M.J. P. A. Veith P.D. Reynolds E.C. In structure and of the type IX secretion 2020; Scholar). These are in the the and are to into the by the PorK These structures to of protein with a of However, the of the to on the structure of their into the or their of In this we on the PorN protein In silico and that the N- and C-terminal regions of PorN are intrinsically disordered. showed that these regions are involved in PorN and PorK the structure of the PorN central by a new and showed that mediates contacts with PorM and effector In a we have that the PorN periplasmic protein of the signal forms and with PorM and PorK (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar). In to we and and extensive no crystal of the PorN the of intrinsically disordered N- and C-terminal which intrinsically disordered regions are more to the protein to with and a domain after with the at a of that the folded domain at and the by that at of PorN of and are The are and for and The the of the The the PorN and to of showed that the PorN folded is in to the of PorN These results that the N- C-terminal involved in To the of the N- and C-terminal regions to PorN we of the N- or These PorN to the and domains of the and by bacterial Our results that and or that the N-terminal region is required for PorN in The PorN to an N-terminal by and and to that and are in to is the in and in that PorN is by the N-terminal intrinsically disordered PorN to with the PorK the PorM periplasmic as well as the T9SS effector (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar, K. Naito M. Yukitake H. H. M. McBride M.J. Nakayama K. A protein secretion system linked to gliding motility and S. A. Scholar, M.S. M. Cascales E. A conserved of Porphyromonas type IX secretion C-terminal secretion signal with the 2018; Scholar, D.G. Veith P.D. Glew M.D. Sato K. Yukitake H. Nakayama K. Reynolds E.C. insights into the PorK and PorN of the Porphyromonas gingivalis type IX secretion Pathog. 2016; Scholar). To information on the role of the N- and C-terminal disordered regions and of the central folded the PorN for their to with PorM, and effector by The of the PorM periplasmic domain the domain and the of T9SS substrates the hemin-binding protein the peptidylarginine deiminase and the T9SS PorU and to the domain and that and with PorK and with In PorN with the of T9SS substrates These results that the PorN C-terminal disordered region is required for the interaction with the PorN central is to contacts with PorM and the to of the of N- and C-terminal intrinsically disordered with the to the and and a at and an model to are present in the this by as and showed that is and However, the of the PorN in the crystal to a or to In of the and of to of and a at 2.0-Å The model of to the structure of by are present in the with the as The of and an of that the are the N-terminal disordered region of is in the and the structure of to the of the is with the crystal that has of that the protein cleaved after which to the crystal and the of the to that of The structure of the PorN folded of an on by by with The is of on and on the with the common to the of the The and of the PorN folded domain are to other that the N- and C-terminal disordered domains are to at A similarity the Protein of the PorN folded domain structure the L. P. in Res. Scholar) any with significant The to the outer membrane which into an of the with the of the PorN with a of the PorN to a of the structures to the by the of similarity with any known structure that the PorN domain represents a new The T9SS is a large complex that delivers substrate proteins into the medium or that them to the cell surface Silva I. Cascales E. Molecular strategies underlying Porphyromonas gingivalis virulence.J. Mol. Biol. 2021; 433: 166836Google Scholar, 11McBride M.J. Bacteroidetes gliding motility and the type IX secretion system.Microbiol. Spectr. 2019; 7https://doi.org/10.1128/microbiolspec.PSIB-0002-2018Google Scholar, 20Veith P.D. Glew M.D. Gorasia D.G. Reynolds E.C. Type IX secretion: The generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers.Mol. Microbiol. 2017; 106: 35-53Google Scholar, 24Lasica A.M. Ksiazek M. Madej M. Potempa J. The type IX secretion system (T9SS): Highlights and recent insights into its structure and function.Front. Cell. Infect. Microbiol. 2017; 7: 215Google Scholar). The recent have to information T9SS and and the structures of T9SS have been we this and the of the P. gingivalis PorN periplasmic with PorN large ring-shaped structures the membrane for which a structure has been D.G. Veith P.D. Glew M.D. Sato K. Yukitake H. Nakayama K. Reynolds E.C. insights into the PorK and PorN of the Porphyromonas gingivalis type IX secretion Pathog. 2016; Scholar). Our that the PorN N- and C-terminal regions are intrinsically the central domain is The structure of the folded domain by and no similarity with any known structure. that PorN (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; bacterial and that dimerization is by the N-terminal PorN dimerization is to as PorN identified by of D.G. Veith P.D. Glew M.D. Sato K. Yukitake H. Nakayama K. Reynolds E.C. insights into the PorK and PorN of the Porphyromonas gingivalis type IX secretion Pathog. 2016; Scholar). that a is of to D.G. Veith P.D. Glew M.D. Sato K. Yukitake H. Nakayama K. Reynolds E.C. insights into the PorK and PorN of the Porphyromonas gingivalis type IX secretion Pathog. 2016; that to 18 PorN are involved in also the of the other PorN domains for interaction with its The C-terminal domain is involved in the interaction with while the folded domain is involved in the interaction with the periplasmic domain of we showed that the PorN folded domain is also involved in the interaction with the T9SS and The model of T9SS substrate that substrates are in the periplasm by the PorM subunit and to the for their secretion across the The the the PorM protein to the with T9SS results in to a central role in the T9SS PorN is also involved in the of the that the the the PorLM energetic and them to the is to that PorN and its in F. a between P. gingivalis PorN and F. johnsoniae that no intrinsically disordered domains are at the with the folded domain is at its with However, an is present after in the and PorN are the of to as the C-terminal disordered domain is present in the as these between PorN and in the assembly of the T9SS complex in the bacterial P. gingivalis and F. However, the a significant of a with the PorN C-terminal the of this in the PorN structure showed that is to the that the and PorN C-terminal regions as interaction with to that with and this of is involved in the in this are in P. gingivalis as of for P. gingivalis in medium with and G. J. A. D. A bacterial system on a signal S. A. Scholar) and for bacterial and protein E. in at or by of or and for this are in The of the N- or or domains and P. gingivalis a with a and by in protein the into by the The is a of the in which a for a and a cleavage by the by of the The the PorN periplasmic domain has been (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar). of PorN the and and and and bacterial the into or the and into and into and into and into and into and and into periplasmic to the and periplasmic and to the domain have been (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar, M.S. E. Cascales E. The of the Porphyromonas gingivalis system the type IX secretion genes the Cell. Infect. Microbiol. 2016; Scholar). the C-terminal domains of RgpB Hbp35 PorU and to the domain are M.S. M. Cascales E. A conserved of Porphyromonas type IX secretion C-terminal secretion signal with the 2018; Scholar). for protein and for bacterial by F. J. A for genes into 2006; as M. A. R. Cascales E. The protein the type secretion system to the cell Microbiol. 75: Scholar). the of to the The of the first has been as for a the or as with to and into the by and bacterial G. J. A. D. A bacterial system on a signal S. A. Scholar) as A. E. The bacterial system on in 2012; Scholar) with the proteins to to the and domains of the of the the proteins into the at for for into of medium with and at of with and and for to at interaction with a protein to the T9SS A. E. The bacterial system on in 2012; Scholar). The in and a is and as (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar) with E. in medium at at of to of the with and for an 18 at by at for in and at and and by The and by at for and the proteins the by a in A The proteins with A with and by in and with the as the that the in medium B. H. Singh M. of proteins and Microbiol. 2001; Scholar). of with for at the to the and by and N-terminal the and proteins on a in at a of an system an and a The and at and the at in with and and and and to in the and the in and after A. A. L. S. M. Cambillau C. of and of a protein the Biol. the for and and in and in with for and for The in a at a to on at the and of to on at the The with and Biol. Scholar) and with P. and of Biol. 2006; Scholar) the for protein Biol. Scholar). of and C. E. P. G. structure with Mol. Biol. 2007; G. C. C. M. and of information in structure in and Biol. Scholar) and in the structure of Biol. Scholar). The model in P. K. for Biol. Scholar) and as model for with A. A. Molecular with Biol. Scholar) to the structure of The of with and of the structure with E. P. C. C. G. of structures with in Biol. and J.J. structure for Biol. and are in and in are for the in are for the in are for the in are for the in are for the in are for the and model in are for the of in in are for the in a new PorN crystal structure and structure have been in the information (25Vincent M.S. Canestrari M.J. Leone P. Stathopoulos J. Ize B. Zoued A. Cambillau C. Kellenberger C. Roussel A. Cascales E. of the Porphyromonas gingivalis type IX secretion Biol. Chem. 2017; Scholar, M.S. M. Cascales E. A conserved of Porphyromonas type IX secretion C-terminal secretion signal with the 2018; Scholar, G. J. A. D. A bacterial system on a signal S. A. Scholar, A. E. The bacterial system on in 2012; Scholar). The that have no of with the of this the of the and for their and for members of the Roussel and Cascales for and E. A. and P. L. E. C. and P. L. E. A. and P. L. I. L. E. and P. L. I. L. E. and P. L. P. L. E. C. review E. A. and P. L. E. C. and A. R. E. C. and A. R. by the the and the E. and P. and the of a to E. C. The of I. L. S. has been by the

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