Melbourne Dental School - Research Publications
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ItemComplementation in trans of Porphyromonas gingivalis Lipopolysaccharide Biosynthetic Mutants Demonstrates Lipopolysaccharide Exchange(American Society for Microbiology, 2021-04-21)Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host. IMPORTANCE: Porphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.
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ItemType B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage(MDPI, 2022-05)The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins.
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ItemProtein Interactome Analysis of the Type IX Secretion System Identifies PorW as the Missing Link between the PorK/N Ring Complex and the Sov Translocon(AMER SOC MICROBIOLOGY, 2022-02)The type IX secretion system (T9SS) transports cargo proteins through the outer membrane of Bacteroidetes and attaches them to the cell surface for functions including pathogenesis, gliding motility, and degradation of carbon sources. The T9SS comprises at least 20 different proteins and includes several modules: the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, the outer membrane Sov translocon, and the cell attachment complex. However, the spatial organization of these modules is unknown. We have characterized the protein interactome of the Sov translocon in Porphyromonas gingivalis and identified Sov-PorV-PorA as well as Sov-PorW-PorN-PorK to be novel networks. PorW also interacted with PGN_1783 (PorD), which was required for maximum secretion efficiency. The identification of PorW as the missing link completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore. IMPORTANCE The T9SS is a newly identified protein secretion system of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum used by pathogens associated with diseases of humans, fish, and poultry for the secretion and cell surface attachment of virulence factors. The T9SS comprises three known modules: (i) the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, (ii) the outer membrane Sov translocon, and (iii) the cell surface attachment complex. The spatial organization and interaction of these modules have been a mystery. Here, we describe the protein interactome of the Sov translocon in the human pathogen Porphyromonas gingivalis and have identified PorW as the missing link which bridges PorN with Sov and so completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing, for the first time, a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore.
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ItemNo Preview AvailableIn situ structure and organisation of the type IX secretion system( 2020-05-14)Abstract The Bacteroidetes type IX secretion system (T9SS) consists of at least 19 components that translocate proteins with a type A or type B C-terminal domain (CTD) signal across the outer membrane. The overall organisation and architecture of this system including how the secretion pore (Sov) interacts with the other components is unknown. We used cryo-electron tomography to obtain the first images of the T9SS including PorK/N rings inside intact Porphyromonas gingivalis cells. Using proteomics, we identified a novel complex between Sov, PorV and PorA and showed that Sov interacts with the PorK/N rings via PorW and a new component PGN_1783. A separate complex comprising the outer membrane β-barrel protein PorP, PorE, and the type B CTD protein PG1035 was also identified. Similarly, the Flavobacterium johnsoniae PorP-like protein, SprF was found bound to the major gliding motility adhesin, SprB. Based on these data, we propose cell surface anchorage for type B CTD proteins to PorP-like proteins and a unique model where the PorK/N rings function as an outer membrane barrier to maintain the close proximity of the translocon to the shuttle and attachment complexes inside the rings, ensuring the harmonized secretion and cell surface attachment of the T9SS substrates.
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ItemThe Role of Treponema denticola Motility in Synergistic Biofilm Formation With Porphyromonas gingivalis(FRONTIERS MEDIA SA, 2019-12-18)Chronic periodontitis has a polymicrobial biofilm etiology and interactions between key oral bacterial species, such as Porphyromonas gingivalis and Treponema denticola contribute to disease progression. P. gingivalis and T. denticola are co-localized in subgingival plaque and have been previously shown to exhibit strong synergy in growth, biofilm formation and virulence in an animal model of disease. The motility of T. denticola, although not considered as a classic virulence factor, may be involved in synergistic biofilm development between P. gingivalis and T. denticola. We determined the role of T. denticola motility in polymicrobial biofilm development using an optimized transformation protocol to produce two T. denticola mutants targeting the motility machinery. These deletion mutants were non-motile and lacked the gene encoding the flagellar hook protein of the periplasmic flagella (ΔflgE) or a component of the stator motor that drives the flagella (ΔmotB). The specificity of these gene deletions was determined by whole genome sequencing. Quantitative proteomic analyses of mutant strains revealed that the specific inactivation of the motility-associated gene, motB, had effects beyond motility. There were 64 and 326 proteins that changed in abundance in the ΔflgE and ΔmotB mutants, respectively. In the ΔflgE mutant, motility-associated proteins showed the most significant change in abundance confirming the phenotype change for the mutant was related to motility. However, the inactivation of motB as well as stopping motility also upregulated cellular stress responses in the mutant indicating pleiotropic effects of the mutation. T. denticola wild-type and P. gingivalis displayed synergistic biofilm development with a 2-fold higher biomass of the dual-species biofilms than the sum of the monospecies biofilms. Inactivation of T. denticola flgE and motB reduced this synergy. A 5-fold reduction in dual-species biofilm biomass was found with the motility-specific ΔflgE mutant suggesting that T. denticola periplasmic flagella are essential in synergistic biofilm formation with P. gingivalis.
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ItemPG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System(PUBLIC LIBRARY SCIENCE, 2016-10-06)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.
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ItemCharacterisation of the Porphyromonas gingivalis Manganese Transport Regulator Orthologue(PUBLIC LIBRARY SCIENCE, 2016-03-23)PgMntR is a predicted member of the DtxR family of transcriptional repressors responsive to manganese in the anaerobic periodontal pathogen Porphyromonas gingivalis. Our bioinformatic analyses predicted that PgMntR had divalent metal binding site(s) with elements of both manganous and ferrous ion specificity and that PgMntR has unusual twin C-terminal FeoA domains. We produced recombinant PgMntR and four variants to probe the specificity of metal binding and its impact on protein structure and DNA binding. PgMntR dimerised in the absence of a divalent transition metal cation. PgMntR bound three Mn(II) per monomer with an overall dissociation constant Kd 2.0 x 10(-11) M at pH 7.5. PgMntR also bound two Fe(II) with distinct binding affinities, Kd1 2.5 x 10(-10) M and Kd2 ≤ 6.0 x 10(-8) M at pH 6.8. Two of the metal binding sites may form a binuclear centre with two bound Mn2+ being bridged by Cys108 but this centre provided only one site for Fe2+. Binding of Fe2+ or Mn2+ did not have a marked effect on the PgMntR secondary structure. Apo-PgMntR had a distinct affinity for the promoter region of the gene encoding the only known P. gingivalis manganese transporter, FB2. Mn2+ increased the DNA binding affinity of PgMntR whilst Fe2+ destabilised the protein-DNA complex in vitro. PgMntR did not bind the promoter DNA of the gene encoding the characterised iron transporter FB1. The C-terminal FeoA domain was shown to be essential for PgMntR structure/function, as its removal caused the introduction of an intramolecular disulfide bond and abolished the binding of Mn2+ and DNA. These data indicate that PgMntR is a novel member of the DtxR family that may function as a transcriptional repressor switch to specifically regulate manganese transport and homeostasis in an iron-dependent manner.
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ItemNo Preview AvailableLysine acetylation is a common post-translational modification of key metabolic pathway enzymes of the anaerobe Porphyromonas gingivalis(ELSEVIER, 2015-10-14)Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a keystone pathogen in the development of the bacterial-associated inflammatory oral disease chronic periodontitis. Although post-translational modifications (PTMs) of proteins are commonly found to modify protein function in eukaryotes and prokaryotes, PTMs such as lysine acetylation have not been examined in P. gingivalis. Lysine acetylation is the addition of an acetyl group to a lysine which removes this amino acid's positive charge and can induce changes in a protein's secondary structure and reactivity. A proteomics based approach combining immune-affinity enrichment with high sensitivity Orbitrap mass spectrometry identified 130 lysine acetylated peptides from 92 P. gingivalis proteins. The majority of these peptides (71) were attributed to 45 proteins with predicted metabolic activity; these proteins could be mapped to several P. gingivalis metabolic pathways where enzymes catalysing sequential reactions within the same pathway were often found acetylated. In particular, the catabolic pathways of complex anaerobic fermentation of amino acids to produce energy had 12 enzymes lysine acetylated. The results suggest that lysine acetylation may be an important mechanism in metabolic regulation in P. gingivalis, which is vital for P. gingivalis survival and adaptation of its metabolism throughout infection. Statement of significance. Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The ability of the pathogen to induce dysbiosis and disease is related to an array of specific virulence factors and metabolic regulation that enables the bacterium to proliferate in an inflamed periodontal pocket. The mechanisms P. gingivalis uses to adapt to a changing and hostile environment are poorly understood and here we show, for the first time, that enzymes of critical metabolic pathways for energy production in this bacterium were acetylated on certain lysine residues. These enzymes were often found catalysing sequential reactions within the same catabolic pathway. The results suggest that lysine acetylation is an important mechanism of metabolic regulation in P. gingivalis vital for its adaptation and proliferation to produce disease.