molecular oral microbiology molecular oral microbiology Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence † V. Friedrich1, C. Gruber2, I. Nimeth1, S. Pabinger3, G. Sekot1,*, G. Posch1, , F. Altmann2, P. Messner1, O. Andrukhov4 and C. Schaffer€ 1 1 Department of NanoBiotechnology, NanoGlycobiology unit, Universitat€ fur€ Bodenkultur Wien, Vienna, Austria 2 Department of Chemistry, Universitat€ fur€ Bodenkultur Wien, Vienna, Austria 3 AIT Austrian Institute of Technology, Health & Environment Department, Molecular Diagnostics, Vienna, Austria 4 Division of Conservative Dentistry and Periodontology, Competence Centre of Periodontal Research, University Clinic of Dentistry, Medical University of Vienna, Vienna, Austria Correspondence: Christina Schaffer,€ Department of NanoBiotechnology, NanoGlycobiology unit, Universitat€ fur€ Bodenkultur Wien, Muthgasse 11, A-1190 Vienna, Austria Tel.: + 43 1 47654 2203; fax: + 43 1 4789112; E-mail: [email protected] *Present address: Baxter AG, Vienna, Austria. †Present address: GlycoVaxyn, Schlieren, Switzerland. Keywords: atomic force microscopy; electron microscopy; inflammatory response; outer membrane vesicles; periodontal pathogen; Tannerella forsythia strain ATCC 43037 versus strain 92A2 Accepted 1 May 2015 DOI: 10.1111/omi.12104 SUMMARY Tannerella forsythia is the only ‘red-complex’ bac- be extracellular, and 39 to originate from the cyto- terium covered by an S-layer, which has been plasm. Eighty proteins contained the Bacteroi- shown to affect virulence. Here, outer membrane dales O-glycosylation motif, 18 of which were vesicles (OMVs) enriched with putative glycopro- confirmed as glycoproteins. Release of pro- teins are described as a new addition to the viru- inflammatory mediators from the human mono- lence repertoire of T. forsythia. Investigations of cytic cell line U937 and periodontal ligament fibro- this bacterium are hampered by its fastidious blasts upon stimulation with OMVs followed a growth requirements and the recently discovered concentration-dependent increase that was more mismatch of the available genome sequence pronounced in the presence of soluble CD14 in (92A2 = ATCC BAA-2717) and the widely used conditioned media. The inflammatory response T. forsythia strain (ATCC 43037). T. forsythia was was significantly higher than that caused by grown anaerobically in serum-free medium and whole T. forsythia cells. Our study represents the biogenesis of OMVs was analyzed by electron first characterization of T. forsythia OMVs, and atomic force microscopy. This revealed OMVs their proteomic composition and immunogenic with a mean diameter of ~100 nm budding off potential. from the outer membrane while retaining the S- layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological repli- INTRODUCTION cates identified 175 proteins. Of these, 14 exhib- ited a C-terminal outer membrane translocation Tannerella forsythia, Porphyromonas gingivalis and signal that directs them to the cell/vesicle surface, Treponema denticola are Gram-negative anaerobes 61 and 53 were localized to the outer membrane that have been identified as major periodontal and periplasm, respectively, 22 were predicted to pathogens (Socransky et al., 1998). Together, they © 2015 The Authors Molecular Oral Microbiology Published by John Wiley & Sons Ltd. 451 Molecular Oral Microbiology 30 (2015) 451–473 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Tannerella forsythia outer membrane vesicles V. Friedrich et al. constitute the so-called ‘red-complex’ consortium that establishing colonization, carrying and transmitting vir- colonizes dental plaque biofilms (Holt & Ebersole, ulence factors into host cells, and modulating host 2005) and is strongly associated with the clinical defense and response (Ellis & Kuehn, 2010). measures of periodontitis, a multifactorial, inflamma- Regarding the ‘red-complex’ consortium, OMVs tory disease of global importance (Darveau, 2010). have so far only been described in detail for P. gingi- According to a more recent model of pathogenesis, valis (Veith et al., 2014), which is generally the best periodontitis is initiated by a synergistic and dysbiotic investigated ‘red-complex’ bacterium. P. gingivalis microbial community rather than by select periodontal OMVs are found in gingival tissues at diseased sites pathogens, such as the ‘red complex’ (Hajishengallis in chronic periodontitis but not at healthy sites (O’Bri- & Lamont, 2012). In this polymicrobial synergy, differ- en-Simpson et al., 2009) and are recognized as ent members or specific gene combinations within the important virulence factors that are produced when community fulfill distinct roles that converge to shape P. gingivalis is part of a polymicrobial biofilm (Zhu and stabilize a disease-provoking microbiota. et al., 2013). The P. gingivalis OMVs can invade host Apart from oral health issues, emerging evidence epithelial cells via an endocytic pathway (Furuta shows a relationship between periodontitis, cardiovas- et al., 2009) and impair cellular functions by the gingi- cular disease, rheumatoid arthritis and other systemic pain-exerted degradation of important receptor pro- chronic diseases (Cullinan et al., 2009; Koziel et al., teins (Veillard et al., 2012; Wilensky et al., 2015). In 2014). For the development of new therapeutic strate- the context of the ‘red complex’, a synergistic effect gies to combat periodontitis, a molecular understand- of P. gingivalis OMVs has become obvious through ing of the mechanisms governing bacterial virulence their enhancement of T. forsythia attachment to epi- is required. Apart from distinct virulence factors that thelial cells (Inagaki et al., 2006). A recent study sup- have been identified for the ‘red-complex’ bacteria ports an interdependence between P. gingivalis (O’Brien-Simpson et al., 2004), outer membrane vesi- virulence factors and T. forsythia, revealing that cles (OMVs), which are also known as integral parts P. gingivalis gingipains influence the composition of of biofilm matrices (Flemming et al., 2007), are polymicrobial biofilms (Bao et al., 2014). emerging as ‘bacterial warfare’ agents in the patho- For T. forsythia, the identification of new virulence genesis of periodontitis. factors (Sharma, 2010) is hampered by its fastidious Generally, OMVs are natural secretion products of growth requirements and the recently discovered mis- Gram-negative bacteria, with an increasing number of match of the available genome sequence pathogens being reported to release OMVs (Unal (92A2=ATCC BAA-2717) and the deposited T. for- et al., 2011; Schertzer & Whiteley, 2013). They are sythia strain (ATCC 43037) (Friedrich et al., 2015). small, spherical particles, usually 20–250 nm in diam- So far, only a few putative virulence factors of T. for- eter, and secreted throughout the bacterial life cycle sythia have been identified. These include trypsin-like and in a wide range of environmental conditions. (Amano et al., 2014) and PrtH (Saito et al., 1997) Although the mechanism of OMV biogenesis is still proteases, the sialidases SiaH (Horstman & Kuehn, poorly understood, studies so far point towards a 2000) and NanH (Thompson et al., 2009; Stafford highly regulated process that is most likely essential et al., 2012), apoptosis-inducing activity (Evans et al., to the cell. Current models suggest that vesiculation 2012), a-D-glucosidase and N-acetyl-b-glucosamini- occurs by budding off from the outer membrane (OM) dase (Hughes et al., 2003), a hemagglutinin (Bomber- at sites where lipoprotein links between the OM and ger et al., 2009), methylglyoxal (Forsberg et al., the peptidoglycan are lost (Kulp & Kuehn, 2010). 1981), a leucine-rich repeat cell surface-associated OMVs are characterized by selective enrichment or and secreted protein BspA (Sharma et al., 1998), the exclusion of specific cargo (Haurat et al., 2011); con- KLIKK proteases (Ksiazek et al., 2015b) such as kari- taining, apart from OM-derived components such as lysin (Karim et al., 2010; Koziel et al., 2010) and lipopolysaccharide (LPS), phospholipids and OM pro- mirolase (Ksiazek et al., 2015a) as well as the S- teins, also periplasmic constituents. OMVs are gain- layer (Sabet et al., 2003). The latter belong to the ing increasing attention as a major mechanism by major class of proteins carrying a C-terminal OM which pathogens attack and interact with host cells. translocation signal known as CTD (C-terminal As such, they have been shown to play roles in domain) (Lee et al., 2006; Sato et al., 2013; Narita 452 © 2015 The Authors Molecular Oral Microbiology Published by John Wiley & Sons Ltd. Molecular Oral Microbiology 30 (2015) 451–473 V. Friedrich et al. Tannerella forsythia outer membrane vesicles et al., 2014; Tomek et al., 2014). Similarly to P. gingi- leukin-6 (IL-6), IL-8, monocyte chemoattractant pro- valis and Treponema denticola (Dashper et al., tein 1 (MCP-1)] from human macrophages and 2011), T. forsythia secretes large amounts of CTD periodontal ligament fibroblasts was determined in proteins by directing them to a type IX secretion sys- comparison to whole T. forsythia cells. Our data sup- tem (T9SS), the presence of which has been demon- port the virulent character of T. forsythia OMVs and strated in T. forsythia only recently (Narita et al., indicate that they are enriched in putative glycopro-