Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32?36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component

Por Secretion System-Dependent Secretion and Glycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study

A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression

Tannerella forsythia is a pathogen implicated in periodontitis, an inflammatory disease of the tooth-supporting tissues often leading to tooth loss. This key periodontal pathogen is decorated with a unique glycan core O-glycosidically linked to the bacterium's proteinaceous surface (S)-layer lattice and other glycoproteins. Herein, we show that the terminal motif of this glycan core acts to modulate dendritic cell effector functions to suppress T-helper (Th)17 responses. In contrast to the wild-type bacterial strain, infection with a mutant strain lacking the complete S-layer glycan core induced robust Th17 and reduced periodontal bone loss in mice. Our findings demonstrate that surface glycosylation of this pathogen may act to ensure its persistence in the host likely through suppression of Th17 responses. In addition, our data suggest that the bacterium then induces the Toll-like receptor 2–Th2 inflammatory axis that has previously been shown to cause bone destruction. Our study provides a biological basis for pathogenesis and opens opportunities in exploiting bacterial glycans as therapeutic targets against periodontitis and a range of other infectious diseases

Prospective Randomized Controlled Trial to Analyze the Effects of Intermittent Pneumatic Compression on Edema Following Autologous Femoropopliteal Bypass Surgery

Background: Patients who undergo autologous femoropopliteal bypass surgery develop postoperative edema in the revascularized leg. The effects of intermittent pneumatic compression (IPC) to treat and to prevent postreconstructive edema were examined in this study. Methods: In a prospective randomized trial, patients were assigned to one of two groups. All patients suffered from peripheral arterial disease, and all were subjected to autologous femoropopliteal bypass reconstruction. Patients in group 1 used a compression stocking (CS) above the knee exerting 18 mmHg (class I) on the leg postoperatively for 1 week (day and night). Patients in group 2 used IPC on the foot postoperatively at night for 1 week. The lower leg circumference was measured preoperatively and at five postoperative time points. A multivariate analysis was done using a mixed model analysis of variance. Results: A total of 57 patients were analyzed (CS 28; IPC 29). Indications for operation were severe claudication (CS 13; IPC 13), rest pain (10/5), or tissue loss (7/11). Revascularization was performed with either a supragenicular (CS 13; IPC10) or an infragenicular (CS 15; IPC 19) autologous bypass. Leg circumference increased on day 1 (CS/IPC): 0.4%/2.7%, day 4 (2.1%/6.1%), day 7 (2.5%/7.9%), day 14 (4.7%/7.3%), and day 90 (1.0%/3.3%) from baseline (preoperative situation). On days 1, 4, and 7 there was a significant difference in leg circumference between the two treatment groups. Conclusions: Edema following femoropopliteal bypass surgery occurs in all patients. For the prevention and treatment of that edema the use of a class I CS proved superior to treatment with IPC. The use of CS remains the recommended practice following femoropopliteal bypass surgery

Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase

Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors to the outer membrane of Gram-negative bacteria. Membrane anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer-membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the bla[subscript NDM] gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies.Kinship Foundation. Searle Scholars ProgramMassachusetts Institute of Technology. Department of Chemistr

Involvement of the Wbp pathway in the biosynthesis of Porphyromonas gingivalis lipopolysaccharide with anionic polysaccharide

The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharide (LPS) molecules, O-LPS and A-LPS. We have recently shown that P. gingivalis strain HG66 lacks A-LPS. Here, we found that introduction of a wild-type wbpB gene into strain HG66 restored formation of A-LPS. Sequencing of the wbpB gene from strain HG66 revealed the presence of a nonsense mutation in the gene. The wbpB gene product is a member of the Wbp pathway, which plays a role in the synthesis of UDP-ManNAc(3NAc)A in Pseudomonas aeruginosa; UDP-ManNAc(3NAc)A is sequentially synthesized by the WbpA, WbpB, WbpE, WbpD and WbpI proteins. We then determined the effect of the PGN-0002 gene, a wbpD homolog, on the biosynthesis of A-LPS. A PGN-0002-deficient mutant demonstrated an A-LPS biosynthesis deficiency. Taken together with previous studies, the present results suggest that the final product synthesized by the Wbp pathway is one of the sugar substrates necessary for the biosynthesis of A-LPS

The Type IX Secretion System: Advances in Structure, Function and Organisation

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

Mass spectrometric analysis of gingival crevicular fluid biomarkers can predict periodontal disease progression

BACKGROUND AND OBJECTIVE: Gingival crevicular fluid has been suggested as a possible source of biomarkers for periodontal disease progression. This paper describes a technique for the analysis of gingival crevicular fluid from individual sites using mass spectrometry. It explores the novel use of mass spectrometry to examine the relationship between the relative amounts of proteins and peptides in gingival crevicular fluid and their relationship with clinical indices and periodontal attachment loss in periodontal maintenance patients. The aim of this paper was to assess whether the mass spectrometric analysis of gingival crevicular fluid may allow for the site-specific prediction of periodontal disease progression. MATERIAL AND METHODS: Forty-one periodontal maintenance subjects were followed over 12 mo, with clinical measurements taken at baseline and every 3 mo thereafter. Gingival crevicular fluid was collected from subjects at each visit and was analysed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Samples were classified based upon pocket depth, modified gingival index (MGI), plaque index and attachment loss, and were analysed within these groups. A genetic algorithm was used to create a model based on pattern analysis to predict sites undergoing attachment loss. RESULTS: Three hundred and eighty-five gingival crevicular fluid samples were analysed. Twenty-five sites under observation in 14 patients exhibited attachment loss of > 2 mm over the 12-mo period. The clinical indices pocket depth, MGI, plaque levels and bleeding on probing served as poor discriminators of gingival crevicular fluid mass spectra. Models generated from the gingival crevicular fluid mass spectra could predict attachment loss at a site with a high specificity (97% recognition capability and 67% cross-validation). CONCLUSIONS: Gingival crevicular fluid mass spectra could be used to predict sites with attachment loss. The use of algorithm-generated models based on gingival crevicular fluid mass spectra may provide utility in the diagnosis of periodontal disease

Lysine acetylation is a common post-translational modification of key metabolic pathway enzymes of the anaerobe Porphyromonas gingivalis

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

Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway

Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis, the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo proteins were deglycosylated using trifluoromethanesulfonic acid and digested with trypsin or proteinase K. The residual modifications were then extensively analyzed by tandem mass spectrometry. The C terminus of each cargo protein was amide-bonded to a linking sugar whose structure was deduced to be 2-N-seryl, 3-N-acetylglucuronamide in P. gingivalis and 2-N-glycyl, 3-N-acetylmannuronic acid in T. forsythia The structures indicated the involvement of the Wbp pathway to produce 2,3-di-N-acetylglucuronic acid and a WbpS amidotransferase to produce the uronamide form of this sugar in P. gingivalis The wbpS gene was identified as PGN_1234 as its deletion resulted in the inability to produce the uronamide. In addition, the P. gingivalis vimA mutant which lacks A-LPS was successfully complemented by the T. forsythia vimA gene; however, the linking sugar was altered to include glycine rather than serine. After removal of the acetyl group at C-2 by the putative deacetylase, VimE, VimA presumably transfers the amino acid to complete the biosynthesis. The data explain all the enzyme activities required for the biosynthesis of the linking sugar accounting for six A-LPS-specific genes. The linking sugar is therefore the key compound that enables the attachment of cargo proteins in P. gingivalis and T. forsythia We propose to designate this novel linking sugar biosynthetic pathway the Wbp/Vim pathway.IMPORTANCE Porphyromonas gingivalis and Tannerella forsythia, two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies