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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ItemCharacterization of the O-Glycoproteome of Flavobacterium johnsoniae(AMER SOC MICROBIOLOGY, 2023-06-27)Flavobacterium johnsoniae is a free-living member of the Bacteroidota phylum that is found in soil and water. It is frequently used as a model species for studying a type of gliding motility dependent on the type IX secretion system (T9SS). O-Glycosylation has been reported in several Bacteroidota species, and the O-glycosylation of S-layer proteins in Tannerella forsythia was shown to be important for certain virulence features. In this study, we characterized the O-glycoproteome of F. johnsoniae and identified 325 O-glycosylation sites within 226 glycoproteins. The structure of the major glycan was found to be a hexasaccharide with the sequence Hex-(Me-dHex)-Me-HexA-Pent-HexA-Me-HexNAcA. Bioinformatic localization of the glycoproteins predicted 68 inner membrane proteins, 60 periplasmic proteins, 26 outer membrane proteins, 57 lipoproteins, and 9 proteins secreted by the T9SS. The glycosylated sites were predominantly located in the periplasm, where they are postulated to be beneficial for protein folding/stability. Six proteins associated with gliding motility or the T9SS were demonstrated to be O-glycosylated. IMPORTANCE Flavobacterium johnsoniae is a Gram-negative bacterium that is found in soil and water. It is frequently used as a model species for studying gliding motility and the T9SS. In this study, we characterized the O-glycoproteome of F. johnsoniae and identified 325 O-glycosylation sites within 226 glycoproteins. The glycosylated domains were mainly localized to the periplasm. The function of O-glycosylation is likely related to protein folding and stability; therefore, the finding of the glycosylation sites has relevance for studies involving expression of the proteins. Six proteins associated with gliding motility or the T9SS were demonstrated to be O-glycosylated, which may impact the structure and function of these components.
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ItemNo Preview AvailableProtein interactome mapping of Porphyromonas gingivalis provides insights into the formation of the PorQ-Z complex of the type IX secretion system(WILEY, 2023-02)Porphyromonas gingivalis is an anaerobic Gram-negative human oral pathogen highly associated with the more severe forms of periodontal disease. Porphyromonas gingivalis utilises the type IX secretion system (T9SS) to transport ∼30 cargo proteins, including multiple virulence factors, to the cell surface. The T9SS is a multiprotein system consisting of at least 20 proteins, and recently, we characterised the protein interactome of these components. Similar to the T9SS, almost all biological processes are mediated through protein-protein interactions (PPIs). Therefore, mapping PPIs is important to understand the biological functions of many proteins in P. gingivalis. Herein, we provide native migration profiles of over 1000 P. gingivalis proteins. Using the T9SS, we demonstrate that our dataset is a useful resource for identifying novel protein interactions. Using this dataset and further analysis of T9SS P. gingivalis mutants, we discover new mechanistic insights into the formation of the PorQ-Z complex of the T9SS. This dataset is a valuable resource for studies of P. gingivalis.
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ItemThe Type IX Secretion System and Its Role in Bacterial Function and Pathogenesis(SAGE PUBLICATIONS INC, 2022-04)Porphyromonas, Tannerella, and Prevotella species found in severe periodontitis use the Type IX Secretion System (T9SS) to load their outer membrane surface with an array of virulence factors. These virulence factors are then released on outer membrane vesicles (OMVs), which penetrate the host to dysregulate the immune response to establish a positive feedback loop of chronic, inflammatory destruction of the tooth's supporting tissues. In this review, we present the latest information on the molecular architecture of the T9SS and provide mechanistic insight into its role in secretion and attachment of cargo proteins to produce a virulence coat on cells and OMVs. The recent molecular structures of the T9SS motor comprising PorL and PorM as well as the secretion pore Sov, together with advances in the overall interactome, have provided insight into the possible mechanisms of secretion. We propose the presence of PorL/M motors arranged in a circle at the inner membrane with bent periplasmic rotors interacting with the PorN protein. At the outer membrane, we envisage a slide carousel model where the PorN protein is driven around a circular track composed of PorK. Cargo proteins are transported by PorN to PorW and the Sov translocon just as slides are rotated to the projection window. Secreted proteins are proposed to then be shuttled along highways consisting of the PorV shuttle protein to an array of attachment complexes distributed around the cell. The cell surface attachment of cargo is a hallmark of the T9SS, and in Porphyromonas gingivalis and Tannerella forsythia, this attachment is achieved via covalent bonding to a linking sugar synthesized by the Wbp/Vim pathway. The cell-surface attached cargo are enriched on OMVs, which are then released from the cell.
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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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ItemTowards defining the outer membrane proteome of Porphyromonas gingivalis(WILEY, 2021-02)Porphyromonas gingivalis is a Gram-negative anaerobic pathogen found in subgingival plaque associated with progressive periodontitis. Proteins associated with the outer membrane (OM) of Gram-negative pathogens are particularly important for understanding virulence and for developing vaccines. The aim of this study was to establish a reliable list of outer membrane associated proteins (Omps) for this organism. Starting with a list of 99 experimentally determined Omps, several bioinformatics tools were used to predict a further 52 proteins, leading to a predicted OM proteome of 151 proteins. The tools used included databases of protein families, prediction of OM β-barrels and structural homology. The list includes 33 T9SS cargo proteins, 43 lipoproteins and 66 OM β-barrel proteins with some overlap between categories. The proteins are discussed both in these structural categories as well as their various functions in OM biogenesis, nutrient acquisition, protein secretion, adhesion and efflux. Proteins that were previously shown to be part of large complexes are highlighted and cross reference is provided to a previous major study of protein localization in P. gingivalis. Finally, proteins were also scored according to their level of conservation within the Bacteroidales taxon. Low scores were shown to correlate with virulence factors and may be predictive of novel virulence factors.
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ItemQuantitative proteomic analysis of the type IX secretion system mutants in Porphyromonas gingivalis(WILEY, 2020-04)Porphyromonas gingivalis is an anaerobic, gram-negative human oral pathogen highly associated with chronic periodontitis. P. gingivalis utilizes the type IX secretion system (T9SS) to transport many of its virulence factors including the gingipains to the cell surface. The T9SS is comprised of at least 16 proteins and the involvement of these 16 proteins in the T9SS has been verified by creating gene deletion mutants in P. gingivalis. These T9SS mutants are regularly utilized to understand how these proteins function together to allow the secretion of the T9SS substrates. We performed label-free quantitative proteomic analysis on the T9SS protein mutants in P. gingivalis to understand the relative abundance of each T9SS component in different mutants. The T9SS components were reduced in abundance in the porK, porL, porM, porN, sov and porT mutants, whereas they were increased in the porE, porU, porV, porZ and porQ mutants. Sov and PorW appear to be the lowest in abundance and PorV the highest amongst all the T9SS components in P. gingivalis wild-type strain. These results are consistent with the proposed role of Sov as the translocation pore in the outer membrane and PorV as the shuttle protein that transports the T9SS substrates between sub-complexes. Together, the label-free quantitative proteomics analyses showed that different T9SS mutants have vastly different abundances of the T9SS components. This knowledge will greatly assist in interpreting the phenotype of the T9SS mutants as well as selecting the right mutant for exploring the role of an individual component.
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ItemThe Type IX Secretion System: Advances in Structure, Function and Organisation(MDPI, 2020-08)The type IX secretion system (T9SS) is specific to the Bacteroidetes phylum. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilises the T9SS to transport many proteins-including its gingipain virulence factors-across the outer membrane and attach them to the cell surface. Additionally, the T9SS is also required for gliding motility in motile organisms, such as Flavobacterium johnsoniae. At least nineteen proteins have been identified as components of the T9SS, including the three transcription regulators, PorX, PorY and SigP. Although the components are known, the overall organisation and the molecular mechanism of how the T9SS operates is largely unknown. This review focusses on the recent advances made in the structure, function, and organisation of the T9SS machinery to provide further insight into this highly novel secretion system.