Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors

Abstract

Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC-MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers

“…As with P. gingivalis , T. forsythia uses the T9SS to secrete virulence factors including surface layer proteins and BspA as well as predicted proteases, Leucine‐rich repeat proteins and an amylase (Sharma, ; Narita et al ., ; Tomek et al ., ; Veith et al ., ). In addition, a group of proteinases involved in virulence known as the KLIKK proteases (Koneru et al ., ) may also be secreted via the T9SS (Veith et al ., ). Unlike P. gingivalis that appears to have an amorphous surface layer composed of T9SS substrates, T. forsythia exhibits a true S‐layer composed of two homologous T9SS substrates, TfsA and TfsB (Higuchi et al ., ; Lee et al ., ) that are important for various virulence traits (Sabet et al ., ; Sakakibara et al ., ).…”

“…As with P. gingivalis, T. forsythia uses the T9SS to secrete virulence factors including surface layer proteins and BspA as well as predicted proteases, Leucine-rich repeat proteins and an amylase (Sharma, 2010;Narita et al, 2014;Tomek et al, 2014;Veith et al, 2015). In addition, a group of proteinases involved in virulence known as the KLIKK proteases may also be secreted via the T9SS .…”

The Type IX Secretion System (T9SS): Highlights and Recent Insights into Its Structure and Function

“…Supporting this, proteomic analysis of the T. forsythia OM identified 13 of 26 proteins bearing the classical CTD, including TfsA, TfsB, and BspA (Tanf_04820), but none of the KLIKK proteases (Veith et al, 2009). Conversely, four KLIKK proteases, forsilysin, miropsin-2 (Friedrich et al, 2015), mirolase (Tanf_00440), and karilysin (Veith et al, 2015), were found in outer membrane vesicles (OMVs), although with a low Mascot score. This discrepancy could be explained by the transient presence of these proteases in the periplasm before they enter the OM translocon of T9SS.…”

Bacterial outer membrane vesicles provide an alternative pathway for trafficking of type III secreted effectors into epithelial cells

“…Although the finding that T3SS associated substrates are contained in OMVs is novel, substrates for other secretion systems have previously been found associated with OMVs. For example, OMVs of the Gram-negative oral pathogen Tannerella forsythia are enriched with substrates of the type IX secretion system (44). Interestingly, Ernst et al recently identified a chaperone-independent secretion pathway for T3SS effectors in the intestinal pathogen Shigella , although the involved pathway was not subjected to detailed analysis (45).…”