PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System.

Jacqueline E Heath,Keith J Cross,Catherine A Butler,Lianyi Zhang,Nor A Nor Muhammad,Christine A Seers,Benjamin Peng,Steven M Cleal,Caroline Moore,Paul D Veith,Nada Slakeski,Stuart G Dashper,Eric C Reynolds,Yu-Yen Chen,Md A Motaleb

doi:10.1371/journal.pone.0164313

Abstract

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.

Highlights

Chronic periodontitis is an inflammatory disease characterised by accretion of a polymicrobial biofilm on the tooth, destruction of the supporting tissues of the teeth and tooth loss [1]
Identification of T9SS components was previously conducted by Sato et al [27], using a genome comparison approach of P. gingivalis and C. hutchinsonii, which possess carboxy-terminal domain (CTD)-family proteins, and B. thetaiotaomicron which does not [27]
To identify additional T9SS components this bioinformatic approach was extended whereby the genomes of twenty-four Bacteroidetes spp. were examined using BLASTp, for the ability to code for proteins which are coded by the P. gingivalis W83 genome

Summary

Introduction

Chronic periodontitis is an inflammatory disease characterised by accretion of a polymicrobial biofilm (subgingival plaque) on the tooth, destruction of the supporting tissues of the teeth and tooth loss [1]. The presence of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola in the subgingival plaque is strongly associated with the clinical indicators of disease [4, 5]. Subgingival plaque composed of greater than 10–15% P. gingivalis cells is a predictor of imminent disease progression [6]. This Gram-negative, black-pigmented, anaerobic bacterium has been described as a keystone pathogen of the disease, which through dysregulation of the local immune response disrupts homeostasis causing dysbiosis and disease progression [7,8,9]. P. gingivalis produces a variety of surface-associated virulence factors implicated in pathogenesis, including lipopolysaccharide (LPS), fimbriae, capsular polysaccharide, haemagglutinin (HagA) and the gingipains [10]. The gingipains are Lys-specific (Kgp) and Arg-specific (RgpA and RgpB) cysteine proteinases, considered to be the bacterium’s major virulence factors responsible for ~85% of its proteolytic activity, and are the most studied [8, 11,12,13,14]

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Results

Conclusion