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Localisation of Outer Membrane Proteins in Treponema denticola by Quantitative Proteome Analyses of Outer Membrane Vesicles and Cellular Fractions Paul D Veith, Michelle D Glew, Dhana G Gorasia, Dina Chen, Neil M O'Brien-Simpson, and Eric C Reynolds J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00860 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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Journal of Proteome Research

Localisation of Outer Membrane Proteins in Treponema denticola by Quantitative Proteome Analyses of Outer Membrane Vesicles and Cellular Fractions

Paul D. Veith1, Michelle D. Glew1, Dhana G. Gorasia1, Dina Chen1, Neil M. O’BrienSimpson1 and Eric C. Reynolds1*

1Oral

Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The

University of Melbourne, Victoria, Australia.

Correspondence: Professor Eric Reynolds, [email protected], +61 3 9341 1547

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ABSTRACT The identification and localisation of outer membrane proteins (Omps) and lipoproteins in pathogenic treponemes such as T. denticola (periodontitis) and T. pallidum (syphilis) has been challenging. In this study, label-free quantitative proteomics using MaxQuant was applied to naturally produced outer membrane vesicles (OMVs) and cellular fractions to identify 1448 T. denticola proteins. Of these, 90 proteins were localised to the outer membrane (OM) comprising 59 lipoproteins, 25 β-barrel proteins and six other putative OMassociated proteins. Twenty-eight lipoproteins were localised to the inner membrane (IM) and 43 proteins were assigned to the periplasm. The signal cleavage regions of the OM and IM lipoprotein sequences were different and may reveal the signals for their differential localisation. Proteins significantly enriched in OMVs included dentilisin, proteins containing leucine-rich repeats and several lipoproteins containing FGE-sulfatase domains. Blue native PAGE analysis enabled the native size of the dentilisin complex and Msp to be determined and revealed that the abundant β-barrel Omps TDE2508 and TDE1717 formed large complexes. In addition to the large number of integral Omps and potentially surface-located lipoproteins identified in T. denticola, many such proteins were also newly identified in T. pallidum through homology, generating new targets for vaccine development in both species.

Key Words: Treponema denticola; Treponema pallidum; outer membrane proteins; bacterial lipoproteins; spirochete; proteome; outer membrane vesicles; protein sorting; localization


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INTRODUCTION Treponemes are Gram-negative, motile, anaerobic spirochetes with characteristic helical morphology, some of which are associated with chronic diseases of humans. T. pallidum is the best studied treponeme and is the causative agent of syphilis 1, while T. denticola is associated with chronic periodontitis together with other bacteria, particularly P. gingivalis and T. forsythia 2. P. gingivalis is considered the major periodontopathogen and T. denticola provides various synergistic roles including nutrient sharing (syntrophy 3), biofilm formation 4, 5

and virulence 6. The virulence factors of T. denticola have been reviewed and include the

major sheath protein (Msp), the chymotrypsin-like protease dentilisin, lipoproteins such as factor H binding protein (FhbB) and periplasmic flagella 7, 8. The cellular architecture of T. denticola has been revealed by cryo-electron tomography and shows a cytoplasm containing cytoplasmic filaments, inner membrane (IM), periplasm containing the flagellar filaments and the outer membrane (OM) 9. Unlike typical Gram-negative bacteria, treponemes and most other spirochetes do not have lipopolysaccharide (LPS), but rather the lipid composition of their outer membrane (OM) includes lipoteichoic acids similar to that found in Gram-positive bacteria 10. This key structural uniqueness of spirochetes may explain the relative fragility of the OM, the difficulty of isolating the OM and the uniqueness of their outer membrane proteins (Omps). Together, these factors have made it difficult to confidently identify and localise Omps. Freeze-fracture EM studies of treponemes show a paucity of trans-membrane Omps in T. pallidum, but an abundance in T. denticola 11-13. In T. pallidum, there has been an extensive search to identify these rare transmembrane Omps and identify other potential OM-associated proteins 14. During the 1980s, the potent lipoprotein immunogens identified in T. pallidum were often assigned to the OM with cell surface exposure making them prime vaccine candidates, however numerous studies have later shown that these lipoproteins are associated



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with the inner membrane (IM) consistent with this spirochete’s immunoevasive properties 15. In electron cryotomography studies of whole T. pallidum cells, densities consistent with the localisation of the major lipoproteins to the periplasmic side of the IM were observed 16, 17. In the lyme disease spirochete, Borrelia burgdorferi, the majority of lipoproteins have been localised to the cell surface, however, several other lipoproteins which were previously localised to the cell surface have now been localised to the IM 18. In T. denticola, the dentilisin complex comprising three lipoproteins has been confirmed to be surface exposed 19, but other lipoproteins such as the ABC transporter substrate-binding protein OppA, previously localised to the cell surface 20 is likely to be localised at the IM based on the IM localisation of OppA orthologs in B. burgdorferi 18 and the IM localisation of other ABC transporter substrate-binding proteins in T. pallidum 14. Of these three spirochetes, the largest number of predicted lipoproteins is 166, found in the T. denticola genome 21. Much further work is required to determine their cellular location. Methods used to localise proteins to the OM or cell surface in spirochetes include immunofluorescence using gentle techniques to ensure the OM is not disrupted 14, 19, 22. In these studies, OM disruption is excluded using control antigens known to be in the periplasm. In B. burgdorferi, surface localisation has been detected by Proteinase K cleavage of surface lipoproteins followed by quantitative mass spectrometry or Western blot analyses performed relative to untreated controls 18. Similarly, the same detection methods were employed to distinguish IM and OM lipoproteins using OM-enriched fractions and OM-deficient fractions 18.

Likewise, a detergent such as Triton X-114 can be used at two different concentrations,

one to solubilize both membranes, and one to solubilize the OM preferentially 23. Although not done in this study, a quantitative proteomic comparison of these two fractions should enable proteins localised to the IM and OM to be distinguished. In this study we utilised


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naturally produced outer membrane vesicles (OMVs) of T. denticola as our enriched OM fraction to aid protein localisation. Gram-negative bacteria naturally produce OMVs by “blebbing” of their OM. OMVs are composed of a single membrane corresponding to the OM of the cell and contains OM proteins and lipids, while the vesicle lumen mainly contains periplasmic proteins 24. OMVs are important virulence factors involved in bacterial adherence, defence against host immunity, and the delivery of toxins 24. The OMVs of T. denticola were previously examined and found to be 50-100 nm in diameter and exhibited a similar protein profile as the OM 25. T. denticola OMVs, likely due to the presence of dentilisin, were able to disrupt cellular tight junctions enabling penetration of epithelial cell monolayers 26. T. denticola OMVs weakly stimulated Toll-like receptors 2 and 4 (TLR2 & TLR4) but activated macrophage inflammasomes and cytokine secretion 27, 28. It is beneficial to study the proteomes of OMVs for several purposes including the characterisation of OMV-based vaccines, identification of virulence factors, OMV biogenesis and protein localization 29. Regarding protein localisation, the advantage of studying OMVs is that they represent a natural OM fraction, that depending on the species, can be essentially free of IM contamination. Our recent studies of the OMV proteomes of P. gingivalis and T. forsythia, for example, demonstrated this approach to be a powerful tool for localising OM proteins 30, 31. In this study we report the proteome of T. denticola OMVs and cells and identify 59 OM-associated lipoproteins and 25 putative β-barrel Omps.



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EXPERIMENTAL PROCEDURES Growth of T. denticola T. denticola ATCC 35405 was cultured in 400 mL batches in Oral Bacteria Growth Media (OBGM) and supplements to late exponential phase (OD650 = 0.3) at 37 °C under anaerobic conditions exactly as described previously 27. Importantly, the rabbit serum component of this medium was filtered through a 10 kDa MWCO ultrafiltration device to avoid overcontamination of the OMVs with serum proteins 27.

Preparation of OMV Samples OMVs were purified exactly as previously published 27. Briefly, T. denticola cells were removed by centrifugation at 8,000 x g for 30 min at 4°C. The culture supernatant was filtered through a 0.22 μm filter and then concentrated through a 100-kDa ultrafilter. The collected concentrate was centrifuged at 100,000 x g for 2 hours at 4°C to yield a crude OMV preparation that was further purified using OptiPrep™ density gradient centrifugation at 150,000 x g for 48 hours at 4°C. Fractions containing the purified OMVs were pooled, washed with 0.01 M phosphate buffered saline, pH 7.4 (PBS), by centrifugation at 150,000 x g for 2 hours at 4°C, resuspended in 0.22 μm filtered PBS and stored at -80°C.

Preparation of Cellular Samples T. denticola cell pellets from above were washed twice in PBS (8,000 g, 4 C, 30 min), resuspended in 20 mL of cold PBS and 100 µL (1/200) proteinase inhibitor cocktail (PIC, Sigma Aldrich) and passed three times through a French Pressure Cell Press (SLM Instruments) at 100 MPa to achieve cell lysis with the pressed sample collected on ice. After the first passage, a further aliquot of 100 µL PIC was added. The pressure cell unit was cooled to 4 C prior to first use and cooled on ice before each subsequent passage. Unbroken 6 ACS Paragon Plus Environment

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cells were removed by centrifugation (6,000 g, 4 C, 15 min) and the supernatant was retained, which was then centrifuged at 40,000 g, 4 C for 30 min. The resulting supernatant was designated the “cell soluble” sample. The pellet was thoroughly resuspended in 5 mL cold PBS by the aid of an ultrasonic processor (model CPX 750, Cole Parmer) using a 3 mm stepped microtip at 21% amplitude for 2 min. The suspension was then aliquoted into 1.5 mL tubes and centrifuged at 40,000 g, 4 C for 20 min. The washes were discarded, and the pellets designated the “cell membrane” samples. Samples were stored at -80 C. The duplicate cell soluble fractions were later fractionated by ultracentrifugation at 160,000 g, 4 C overnight resulting in the “soluble supernatant” and “soluble pellet” fractions.

SDS-PAGE and In-gel Digestion Proteins in the various fractions were either used directly or were first precipitated with 12% trichloroacetic acid and the pellet washed with ice-cold acetone. All samples were solubilised and denatured by heating in LDS sample solution and 50 mM dithiothreitol at 100 C for 5 min. SDS-PAGE was conducted using NuPAGE Novex 10% Bis-Tris gels with MOPS running buffer (Thermo Fisher Scientific Australia). Gels were run for approximately 6 min at 126 V and stained with Coomassie Blue G250. The samples were excised from each gel lane in one band from the well bottom to the tracking dye. In-gel digestion using trypsin was performed after reduction with dithiothreitol and alkylation with iodoacetamide as previously published 32.

Proteinase K Treatment of T. denticola T. denticola were grown to late-logarithmic phase as described above and gently harvested by centrifugation at 1000 g for 5 min. The pelleted cells were washed 2X with PBS containing 5 mM MgCl2 (PBS-MgCl2) and pelleted as above. Cells were then resuspended in PBS-MgCl2, 7 ACS Paragon Plus Environment


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adjusted to an OD650nm of 1.0 and divided into two tubes. The cells in one tube were lysed by three passages through a French Press cell as described above except that no proteinase inhibitors were used. Equal amounts of cells and lysed cells were then treated with either proteinase K (200 µg/mL) or with water (control) in triplicate for 1 h at room temperature as previously published 18. A portion of each sample was also incubated for 4 h at the same temperature and all reactions were stopped by adding phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 5 mM. Aliquots of cells and cell lysates were mixed with SDSPAGE sample buffer containing 50 mM DTT, boiled for 5 min and subjected to SDS-PAGE and in-gel digestion as described above. Due to minimal digestion after 1h, only data for the 4 h digestions are shown in the results.

Blue Native (BN)-PAGE of T. denticola OMVs After storage at -80°C in PBS the OMVs were pelleted by centrifugation at 170,000 x g for 2 hours at 8°C and the OMV pellet solubilized in 1 × NativePAGE™ sample buffer containing 1% (w/v) n-dodecyl-β-D-maltoside (DDM, Sigma), 20 mM Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (Sigma) and 10 μL/mL protease inhibitor cocktail (PIC, Sigma) as previously published 33. The OMV lysate was then washed once in 1 x NativePAGE™ sample buffer containing 0.5% (w/v) DDM and 10 μL/mL PIC using an Amicon Ultracel100K centrifugal filter (Millipore) to remove excess lipids and small proteins 34. Two dimensional (2D) BN-PAGE was performed as previously described 33, 34.

LC-MS/MS Analysis All samples were analysed by LC-MS/MS using a Q Exactive Plus Orbitrap Mass Spectrometer coupled to an Ultimate 3000 Ultra-High Performance Liquid Chromatography (UHPLC) system (Thermo Fisher Scientific, San Jose, CA). Buffer A was 0.1% (v/v) formic


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acid (FA), 2% (v/v) ACN in water, and buffer B was 0.1% (v/v) FA in ACN. Sample volumes of 5 µL were loaded onto a PepMap C18 trap column (75 µM i.d x 2 cm, 3 µM, 100 Å, Thermo Fisher Scientific) and desalted at a flow rate of 2 µL min-1 for 15 min using buffer A. The samples were then separated through a PepMap 100 C18 analytical column (75 µM i.d x 15 cm, 2 µM, 100 Å, Thermo Fisher Scientific) at a flow rate of 300 nL min-1, with a step gradient of solvent B consisting of 2% to 10% in 1 min, from 10% to 35% in 50 mins, from 35% to 60% in 1 min and from 60% to 90% in 1 min. For analysis of the whole cell lysates and proteinase K digested samples, the gradient was extended to 90 min. The spray voltage was set at 1.8 kV and the temperature of the ion transfer tube was 250°C. The S-lens was set at 50%. The full MS scans were acquired over a m/z range of 300-2000, with a resolving power of 70,000, and an automatic gain control (AGC) target value of 3.0x106 and a maximum injection time of 30 ms. Higher-energy collisional dissociation MS/MS scans (HCD) were acquired at a resolving power of 17,500, AGC target value of 5.0x104, maximum injection time of 120 ms, isolation window of m/z 1.4 and NCE of 25% for the top 15 most abundant ions in the MS spectra. The exclusion time was set to 90 s. Peaklist files (mgf format) for Mascot searching were created using RawConverter v1.1.0.19 with the “select monoisotopic m/z in DDA” option employed and charge states from 2+ to 6+ selected 35.

Protein Identification Proteins were identified by MS/MS ions search using Mascot v2.6 (Matrix Science, UK) against a T. denticola ATCC 35405 protein sequence database obtained from JCVI in May 2005 (originally TIGR). This database was missing many proteins corresponding to genes that were annotated as pseudogenes or having frame-shift mutations. Since many of these proteins were known to be produced their sequences were obtained by translating their



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respective gene sequences. To the 2786 T. denticola protein sequences, the sequence of common contaminants including trypsin, human keratins and abundant rabbit serum proteins were added for a total of 2874 sequences. Search parameters were as follows. Enzyme = trypsin, MS tolerance = 5 ppm, MS/MS tolerance = 0.2 Da, missed cleavages = 2, fixed modifications = carbamidomethyl (Cys), optional modifications = oxidation (Met). Proteins were considered identified when the protein score was > 25 and at least two distinct peptides were identified. Peptides were considered identified when their Mascot ions scores were >15 (p 0.4, 114 proteins exhibited a quantifiable SP/SS result. These included 21 β-barrel Omps and 57 OM-lipoproteins. Of these, all the β-barrel Omps and 41 of the OM-lipoproteins exhibited an SP/SS >3.0, while only slightly lower down on the SP/SS-sorted list were found


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periplasmic binding proteins such as TDE0143, TDE0748 and TDE2234 demonstrating that an SP/SS cut-off of 3.0 was useful for discriminating Omps from periplasmic proteins (Table S2). Using the SP/SS cut-off of 3.0, six additional proteins (TDE0752, TDE0832, TDE0889, TDE0902, TDE1269 and TDE1984) were tentatively localised to the OM (Table S2). Similarly, 40 OMV proteins exhibiting an SP/SS ratio of < 1.3 together with three additional periplasmic binding proteins were tentatively localised to the periplasm. In OMVs, these 43 proteins are likely to reside in the lumen.

Identification of Proteinase K Sensitive Proteins Since OM-lipoproteins can be located on either side of the OM, they were probed using Proteinase K digestion of both whole cells and lysed cells in triplicate followed by quantitation with MaxQuant. The ratios LFQ (treated) / LFQ (untreated) were calculated for each protein where a ratio of 1 theoretically means no cleavage and a low ratio means extensive cleavage. By definition, ratios > 1 are theoretically not possible. However, since MaxQuant normalised the data, the average ratio becomes close to 1 and the highest ratios represent uncleaved proteins. For whole cells after 4h digestion, a distribution plot of all ratios revealed a normal distribution centred around a ratio of 1.0 (Figure 4). The distribution of potentially surface exposed proteins (lipoproteins and OM β-barrel proteins) was similar to the overall distribution (data not shown). Since the distribution was symmetrical and sharp it was concluded that all proteins were resistant to proteinase K under the conditions employed. The distribution simply reflects the errors associated with measuring the protein abundance. Serving as an internal control, the residual rabbit serum proteins from the growth medium typically exhibited ratios of less than 0.2 confirming that the Proteinase K was able to extensively cleave sensitive proteins (data not shown). Treatment of lysed cells caused extensive degradation reflected by a non-symmetrical distribution (Figure 4). After manual



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normalisation to align the highest ratio peak with a ratio of 1, it was found that only 25% of proteins exhibited a ratio greater than 0.7 (Figure 4, Table S1). The β-barrel Omps remained relatively resistant to cleavage with 80% of those quantifiable exhibiting a ratio greater than 0.7. The lipoproteins were more sensitive to cleavage with 50% exhibiting a ratio greater than 0.7. Unfortunately, there was no significant difference between those lipoproteins localised to the OM and IM (Table S2).

Figure 4. Proteinase K digestion of proteins in lysed and unlysed cells. Lysed or unlysed cells were treated with proteinase K for 4 h and subjected to proteomic analysis and quantification with MaxQuant in triplicate. As the unlysed cells were resistant to proteinase K, the LFQ ratios centred around 1.0 (no change). The LFQ ratios calculated for proteins from lysed cells needed further normalisation such that the peak of resistant proteins could be centred on a ratio of 1.0. The y-axis shows the number of proteins quantified within ratio intervals of 0.05.

BN-PAGE To identify potential complexes in the OMVs, OMVs were solubilised in DDM and separated by BN-PAGE followed by a second dimension of SDS-PAGE (Figure 5). The visible spots 22 ACS Paragon Plus Environment

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were excised and identified by LC-MS/MS (Table 5, Table S3, Figure S2). Discrete complexes observed included Msp (spot L, >480 kDa), the dentilisin complex (spots C, D, E, >370 kDa), TDE2508 (spot M, >650 kDa), TDE2735 (spot U, ~300 kDa), TDE2673 (spot Q, >120 kDa) and TDE1717 (spot R, ~240Da).

Figure 5. Coomassie stained 2D BN-PAGE of T. denticola OMVs. Proteins were identified by gel excision of the Coomassie stained protein spot and LC-MS/MS (Table 5, Figure S2, and Table S3). Proteins identified (locus tag) within the same spot were designated a letter or letter with number and listed in Table 5. Dentilisin protease-related proteins are shown in red coloured type, ABC transporter periplasmic substrate-binding proteins are shown in blue, predicted β-barrel outer membrane proteins are shown in green, all other proteins are shown in black.



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DISCUSSION Very few proteomic studies of Treponema denticola have been reported, and this is the first to analyse the OMV proteome. Since the best studied treponeme, Treponema pallidum cannot be grown readily in-vitro, it is not practical to study its natural ability to produce OMVs or to study their protein contents. Borrelia burgdorferi produces natural OMVs and a limited proteomic study of these resulted in the identification of 15 proteins 44. Hence this is the first proteomic study of naturally produced treponemal OMVs and to our knowledge the first indepth proteomic study of natural spirochetal OMVs. In our studies of OMVs produced by two other periodontal pathogens, P. gingivalis and T. forsythia the OMV preparations consisted almost entirely of OM-associated proteins and periplasmic proteins 30, 31. In contrast, in this study, 41% of the OMV proteins identified did not exhibit a putative signal peptide and most of these are therefore likely to be cytoplasmic proteins. The identification of such proteins in OMV preparations is very common, despite the use of rigorous OMV purification regimes 29. Since these cytoplasmic proteins were generally amongst the most abundant cellular proteins, their presence in the OMV preparations is consistent with the spontaneous production of heterogenous vesicles during cell lysis 45. While it is feasible that some proteins without classical signal peptides have been selectively packaged into OMVs, their number would be relatively few. The abundance of cytoplasmic proteins within the OMV preparations was low (8%) with only one (TDE0744, thioredoxin) present in the top 40 most abundant proteins. Since the level of cellular contamination was low, it was possible to show enrichment of certain proteins in the OMVs. These enriched proteins are highly likely to be genuine OMV proteins either associated with the vesicle membrane or packaged inside the vesicle lumen.


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The cell surface located dentilisin lipoprotein complex comprising dentilisin (TDE0762, PrtP), and two accessory proteins (TDE0761, PrcA and TDE0760, PrcB) 19 was significantly enriched in the OMVs (Table 1, Figure 2). PrcA is known to be cleaved into two fragments named PrcA1 and PrcA2. In a non-continuous 2D BN-PAGE experiment, the dentilisin complex was found to migrate at a similar native MW as Msp. Furthermore, PrcA2 was only surface accessible in a mutant lacking Msp suggesting that these two proteins may interact 19. In our study, the 2D BN-PAGE experiment was continuous allowing the resolution of separate Msp and dentilisin complexes solubilised in DDM detergent (Figure 5), however this does not rule out the possibility of an interaction between Msp and dentilisin invivo. Dentilisin is a chymotrypsin-like proteinase able to degrade several important host proteins and has been strongly linked to the virulence of T. denticola in animal models of infection 7, 8, 19, 46. Since dentilisin has been shown to be located on the surface of T. denticola cells it is also expected to be on the surface of the OMVs. OMVs therefore can be expected to be highly virulent particles. The other enriched proteins are of unknown function but include several FGEsulfatase domain proteins and three Leu-rich proteins including LrrA (TDE2258) which appears to be involved in coaggregation with other bacteria and colonisation of host tissues 47.

Known FGE-sulfatase domain proteins are redox active enzymes and in eukaryotes

generally convert a Cys residue in the active site of a sulfatase enzyme into an oxoalanine (formylglycine) residue 48. Leu-rich proteins are frequently involved in protein-protein interactions and if present on the surface of OMVs may be involved with host interactions to aid colonisation, internalisation or other interactions. Most of the enriched proteins were predicted lipoproteins and it is interesting to speculate that most of these are present on the OMV surface since the outer surface of the OMVs has increased surface area relative to the



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cell while the luminal surface of the vesicle membrane has decreased surface area making enrichment at this latter locale less likely. Known surface lipoproteins of P. gingivalis such as HmuY and IhtB were also highly enriched in OMVs giving some precedent to this idea 31. Amongst the OMV-enriched proteins, TDE1148 and TDE1149 were alone in not having a predicted N-terminal signal peptide suggesting they may be packaged into OMVs via a specialised pathway. Amongst treponemal species and strains whose sequences are publicly available, these proteins are unique to a small number of T. denticola strains. The genetic locus for these proteins contains several phage-related proteins which may explain their uniqueness. No other proteins from this locus were identified in OMVs or the cellular fractions. The proteins strongly depleted in OMVs were also mostly lipoproteins including many solute-binding proteins. The depletion of proteins containing the OmpA domain is readily explained by their known ability to bind to peptidoglycan 49. Outer membrane proteins tethered to periplasmic peptidoglycan are not free to be released into budding vesicles as previously documented 31, 50. The retention of the two LolA-like proteins in the cell can be explained by their expected function of lipoprotein sorting between the inner and outer membranes 51, a function not relevant in OMVs. Localisation of OM-associated proteins in spirochetes has proven to be very difficult, especially in T. pallidum where the OM is particularly fragile and special methods are required for immunolocalization 22. However, even with T. denticola, immuno-detection of cell surface antigens requires the use of periplasmic markers as a negative control 19. Previous localisation of antigens to the cell surface without careful controls should be interpreted with caution. Among these are various T. pallidum antigens 52, 53, OppA from T. denticola and B. burgdorferi 20, 54 and other B. burgdorferi lipoproteins (see references in Table 1 of 18). Other methods besides immunolocalization used for OM localisation in spirochetes usually rely on


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a fractionation method such as detergent extraction or gradient centrifugation. The problem however has been that spirochetes appear to harbour an abundance of inner membrane lipoproteins which are highly antigenic, and at least some of these always contaminate OM fractions. This is especially true of T. pallidum since it has a paucity of Omps 55. The isolation of OMs in high purity is also challenging for true Gram-negative bacteria (as opposed to spirochetes) and tends to work best for a subset but not all OM-associated proteins 32. The use of OMVs for protein localisation is advantageous because in many bacteria it is the cleanest “OM fraction” that can be produced. In Bacteroidales species such as P. gingivalis, T. forsythia and B. fragilis, naturally produced OMVs can be isolated with purities up to 99% (e.g. only OM-associated and periplasmic proteins) and have been instrumental in the localisation of proteins to the OM and periplasm 30, 31, 56-58. OMVs derived from Proteobacteria tend to be less pure, but still have utility in localisation experiments 29. In this study, the T. denticola OMVs were pure enough to identify 59 OM lipoproteins and provide an experimental basis for predicting 25 β-barrel proteins and a further six putative Omps. Of the 59 OM lipoproteins, 15 have homologs in T. pallidum including Tp0453 which has already been localised to the inner leaflet of the OM 59, and the Yfio OM lipoprotein which is involved in the targeting and assembly of β-barrel Omps 60. Of the remaining 13, some may be good candidates for having surface exposure, a highly sought-after property in this spirochete. Of the 28 IM lipoproteins, MglB (TDE2217 61), TmpC (TDE1950 62) and peptide transport systems involving OppA (TDE1071 20) and DppA (TDE1072 63) have been characterised in T. denticola and 10 homologs of the IM lipoproteins were found in T. pallidum including five that have been characterised to our knowledge (Table S2 64-69). In E. coli, lipoproteins are sorted between IM and OM locations by the presence of an Asp residue in the +2 position relative to the signal peptide cleavage site. The Asp residue serves as an IM



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retention signal 70. For T. denticola, the cleavage sequence logos for IM and OM lipoproteins were very distinct suggesting that residues in the +2 to +5 positions may be involved in sorting (Figure 3). It would be of considerable interest to test this prediction by creating sitedirected mutants designed to direct IM lipoproteins to the OM and vice versa. Proteinase K has been employed in B. burgdorferi to elegantly identify surface exposed lipoproteins 18. However, when using the same (1 h) or harsher (4 h) digestion conditions with T. denticola we were unable to detect cleavage of surface exposed proteins. T. denticola surface proteins may be designed to resist proteolytic attack in order to withstand the proteolytic environment in which they reside. This includes the abundance of dentilisin on the cell surface of T. denticola, and potentially the very abundant cell surface proteinases (gingipains) of P. gingivalis since these two species can thrive in close proximity in dualspecies biofilms 4. Proteinase K treatment of lysed cells resulted in extensive cleavage of most T. denticola proteins. While OM β-barrel proteins tended to maintain their resistance to cleavage, many other proteins including some cytoplasmic proteins and IM lipoproteins were also resistant, severely limiting the utilisation of this technique for localisation purposes. Potentially, others can mine the data and gain some insight into the structural robustness of their protein(s) of interest. Furthermore, the digestion of surface proteins may work better under different conditions such as the use of a higher temperature or the use of different proteinases. In this study we listed 25 proteins likely to form β-barrels in the OM (Table 4). Of these, only the major sheath protein TDE0405 (Msp or MOSP) has previously been shown to form a β-barrel 71. The C-terminal domain can form trimeric OM β-barrels that were modelled to consist of 10 β-strands per monomer 72 explaining its previously detected surface exposure and porin activity 19, 73. Msp forms SDS-resistant trimers 71, 74 and recombinant Msp solubilised in DDM forms a smear from ~150-400 kDa on BN-PAGE 71. However native


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Msp, also solubilised in DDM migrates to ~480-580 kDa (Figure 5) suggesting that Msp may form higher order complexes in-vivo which are reduced to trimers in the presence of SDS. Based on conserved domain searching (Pfam), two further proteins, TDE1717 and TDE2601(BamA) were predicted to be β-barrels (Table 4), while TDE2117 was likely to be an OM β-barrel based on homology to TP0865 which was predicted to form an OM β-barrel by several prediction algorithms 14. The remaining 21 candidate OM β-barrel proteins in Table 4 required structural homology modelling or -strand analysis for their prediction. Their location in the OM was further validated by their high SP/SS ratios. Strong structural homology matches were obtained to TolC (TDE2285) which forms a conduit through the OM and periplasm for Type I protein secretion or small molecule excretions including multidrug efflux 75; a second BamA-like protein (TDE0688) including the OM β-barrel domain but only one POTRA domain; three porins (TDE0308, TDE0467, TDE2011); two TbuX-related proteins of the FadL family (TDE1884, TDE2117) that usually form 14-stranded barrels 76; a collection of small, usually 8-stranded barrels best matching to OmpA (TDE0014), OmpW (TDE1717, TDE2674) or OprG (TDE2673); and larger barrels best matching to Wzi (TDE1231, TDE1234) or an alginate export protein (TDE1848). In addition to the known homologs in T. pallidum for BamA (TP_0326, 77), Msp (TP_0897, 71) and TDE2117 (TP_0865), five further homologs, namely TP_0733, TP_0856, TP_0548, TP_0859 and TP_0966 were identified that should be considered as rare outer membrane protein candidates in T. pallidum. TDE2508 was predicted to be an OM β-barrel (Table 4, Figure S1) and was found to form the largest identified complex of ~600 kDa (Figure 5). TDE2508 was previously found to form a complex by chemical crosslinking 78. The crosslinking produced a ladder of uniform appearance on SDS-PAGE most consistent with a homotypic complex. The absence



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of any apparent spots aligning with TDE2508 in our 2D BN-PAGE experiment is consistent with this finding. In conclusion, the quantitative proteomic analyses of OMVs and cellular fractions combined with bioinformatic data have resulted in a large number of proteins to be newly localised to the OM of T. denticola. Figure 6 shows a schematic localisation of many Omps of interest along with abundant IM lipoproteins.

Figure 6. Schematic of T. denticola OM and IM showing proteins of interest. OM lipoproteins are shown in green and may be located on either side of the membrane. The dentilisin complex (TDE0760-0762) and FhbB have been localised to the cell surface by others 19, 79. HbpA&B (TDE2055, TDE2056) are likely to be on the surface to bind heme 80, 81 and TDE0730-TDE0732 and TDE2735 are possibly on the surface due to their significant enrichment in OMVs (Table 1). TDE1663 is likely to be on the inside of the OM since it contains the OmpA peptidoglycan motif. TDE2147 shows similarity to the BamD lipoprotein which interacts with BamA. OM β-barrel proteins are shown as light blue rectangles and include Msp (TDE0405), TDE2508, BamA (TDE2601), TolC (TDE2285) and the abundant small barrels, TDE1717 and TDE2673. Seven abundant IM lipoproteins are shown in orange, three of them are part of known ABC transport systems involving the solute binding lipoprotein (orange), permease (dark blue) and ATP-binding protein (purple). For clarity, the periplasmic flagella and peptidoglycan layer were omitted.

ASSOCIATED CONTENT Supporting Information 30 ACS Paragon Plus Environment

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Figure S1. Manual prediction of β-strands in the C-terminal region of putative Omps after multiple alignment. Figure S2. Identification of T. denticola OMV proteins separated by 2D BN-PAGE. Table S1. Summary of Mascot Data for all samples Table S2. Analysis of OMV proteins quantified by MaxQuant Table S3. MS data for all BN-PAGE identified proteins

AUTHOR INFORMATION *Corresponding author: E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This research was supported by the Australian Government Department of Industry, Innovation and Science Grant ID 20080108. The authors thank Mr William Singleton for the provision of cells and Dr Jessica Cecil for the provision of both cells and purified OMV samples. The authors thank Dr Shuai Nie, Dr Ching-Seng Ang and Dr Nicholas Williamson for the acquisition of Orbitrap LC-MS/MS data and their technical support through the Mass



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Spectrometry and Proteomics Facility at Bio21 Institute, The University of Melbourne, Australia.

ABBREVIATIONS BN: blue native; DDM: n-dodecyl-β-D-maltoside; IM: inner membrane; Omp: outer membrane protein; OM: outer membrane; S/M: iBAQ ratio of cell soluble sample / cell membrane sample; SP/SS: iBAQ ratio of soluble pellet sample / soluble supernatant sample


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(62) Abiko, Y.; Nagano, K.; Yoshida, Y.; Yoshimura, F. Characterization of Treponema denticola mutants defective in the major antigenic proteins, Msp and TmpC. PLoS One 2014, 9, e113565. (63) Asai, T.; Okamoto-Shibayama, K.; Kikuchi, Y.; Ishihara, K. Characterization of a novel potential peptide import system in Treponema denticola. Microb. Pathog. 2018, 123, 467-472. (64) Stamm, L. V.; Hardham, J. M.; Frye, J. G. Expression and sequence analysis of a Treponema pallidum gene, tpn38(b), encoding an exported protein with homology to T. pallidum and Borrelia burgdorferi proteins. FEMS Microbiol. Lett. 1996, 135, 57-63. (65) Brautigam, C. A.; Deka, R. K.; Liu, W. Z.; Norgard, M. V. Crystal stuctures of MglB-2 (TP0684), a topologically variant d-glucose-binding protein from Treponema pallidum, reveal a ligand-induced conformational change. Protein Sci. 2018, 27, 880-885. (66) Brautigam, C. A.; Deka, R. K.; Ouyang, Z.; Machius, M.; Knutsen, G.; Tomchick, D. R.; Norgard, M. V. Biophysical and bioinformatic analyses implicate the Treponema pallidum Tp34 lipoprotein (Tp0971) in transition metal homeostasis. J. Bacteriol. 2012, 194, 6771-6781. (67) Brautigam, C. A.; Deka, R. K.; Schuck, P.; Tomchick, D. R.; Norgard, M. V. Structural and thermodynamic characterization of the interaction between two periplasmic Treponema pallidum lipoproteins that are components of a TPR-protein-associated TRAP transporter (TPAT). J. Mol. Biol. 2012, 420, 70-86. (68) Deka, R. K.; Brautigam, C. A.; Yang, X. F.; Blevins, J. S.; Machius, M.; Tomchick, D. R.; Norgard, M. V. The PnrA (Tp0319; TmpC) lipoprotein represents a new family of bacterial purine nucleoside receptor encoded within an ATP-binding cassette (ABC)like operon in Treponema pallidum. J. Biol. Chem. 2006, 281, 8072-8081.



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(69) Hansen, E. B.; Pedersen, P. E.; Schouls, L. M.; Severin, E.; van Embden, J. D. Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli. J. Bacteriol. 1985, 162, 1227-1237. (70) Narita, S. I.; Tokuda, H. Bacterial lipoproteins; biogenesis, sorting and quality control. Biochim. Biophys. Acta 2017, 1862, 1414-1423. (71) Anand, A.; Luthra, A.; Edmond, M. E.; Ledoyt, M.; Caimano, M. J.; Radolf, J. D. The major outer sheath protein (Msp) of Treponema denticola has a bipartite domain architecture and exists as periplasmic and outer membrane-spanning conformers. J. Bacteriol. 2013, 195, 2060-2071. (72) Puthenveetil, R.; Kumar, S.; Caimano, M. J.; Dey, A.; Anand, A.; Vinogradova, O.; Radolf, J. D. The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola. Sci. Rep. 2017, 7, 13260. (73) Egli, C.; Leung, W. K.; Muller, K. H.; Hancock, R. E.; McBride, B. C. Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola. Infect. Immun. 1993, 61, 1694-1699. (74) Fenno, J. C.; Wong, G. W.; Hannam, P. M.; Muller, K. H.; Leung, W. K.; McBride, B. C. Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp. J. Bacteriol. 1997, 179, 1082-1089. (75) Hinchliffe, P.; Symmons, M. F.; Hughes, C.; Koronakis, V. Structure and operation of bacterial tripartite pumps. Annu. Rev. Microbiol. 2013, 67, 221-242. (76) van den Berg, B. The FadL family: unusual transporters for unusual substrates. Curr. Opin. Struct. Biol. 2005, 15, 401-407.


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(77) Luthra, A.; Anand, A.; Hawley, K. L.; LeDoyt, M.; La Vake, C. J.; Caimano, M. J.; Cruz, A. R.; Salazar, J. C.; Radolf, J. D. A homology model reveals novel structural features and an immunodominant surface loop/opsonic target in the Treponema pallidum bama ortholog TP_0326. J. Bacteriol. 2015, 197, 1906-1920. (78) Abiko, Y.; Nagano, K.; Yoshida, Y.; Yoshimura, F. Major membrane protein TDE2508 regulates adhesive potency in Treponema denticola. PLoS One 2014, 9, e89051. (79) McDowell, J. V.; Frederick, J.; Miller, D. P.; Goetting-Minesky, M. P.; Goodman, H.; Fenno, J. C.; Marconi, R. T. Identification of the primary mechanism of complement evasion by the periodontal pathogen, Treponema denticola. Mol. Oral Microbiol. 2011, 26, 140-149. (80) Xu, X.; Holt, S. C.; Kolodrubetz, D. Cloning and expression of two novel hemin binding protein genes from Treponema denticola. Infect. Immun. 2001, 69, 4465-4472. (81) Xu, X.; Kolodrubetz, D. Construction and analysis of hemin binding protein mutants in the oral pathogen Treponema denticola. Res. Microbiol. 2002, 153, 569-577.



Page 44 of 51

Table 1: Proteins and protein groups most significantly enriched in the OMV fraction, organised according to genetic locus or functional group Locus Tag

Protein description

TDE0760-62

Dentilisin and associated proteins

N-terminal signal peptide yes (all LP)

TDE0730-33

Unknown function

yes (3 LP)

TDE2673-74 TDE1660, TDE2258 TDE2735, TDE2737 TDE0571, TDE0572 TDE0833, TDE1232, TDE2101 TDE2269, TDE2495 TDE2515, TDE2714 TDE2116-17 TDE0939-41 TDE1148-49 TDE1489-90 TDE1693 TDE1234 TDE2567

Predicted β-barrel Omps Leu-rich repeat proteins (LRR_5)

yes yes (all LP)

FGE-sulfatase domain proteins

yes (all LP)

Unknown, Predicted β-barrel Omp Putative OM lipoproteins Unknown function Predicted β-barrel Omps Putative OM lipoprotein Predicted β-barrel Omp Predicted β-barrel Omp

yes yes (all LP) no yes yes (LP) yes yes

OMV enrichment Ratios 3.8, 6.4, 10.6 3.4, 5.7, 8.2, 8.6 2.9, 11.3 0.95, 3.4, 3.6, 5.6 0.5, 0.8, 1.8, 2.6, 3.1, 3.9, 3.9, 5.6, 6.1

Combined p-Value

3.0, 5.6 2.6, 3.2, 4.3 3.2, 3.6 8.4, 2.7 6.4 6.3 2.5

0.035 0.021 0.077 0.021 0.035 0.003 0.015

0.009 0.047 0.053 0.028 0.002


Page 45 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2: Proteins and protein groups most significantly depleted in the OMV fraction, organised according to genetic locus or functional group Locus Tag

Protein description

TDE0398, TDE0985, TDE1071, TDE1072, TDE1273, TDE2629 TDE0758, TDE2217

peptide ABC transporter, peptidebinding proteins (SBP_bac_5), lipoproteins only Periplasmic Binding Proteins (lipoproteins only) Treponemal membrane proteins OmpA family proteins

TDE2432, TDE2433 TDE0664, TDE1663, TDE2028 TDE0468, TDE1849 TDE1511 TDE1950 TDE2699 TDE1658 TDE1669

LolA-like proteins Putative IM lipoprotein (iron transport) TmpC lipoprotein putative antigen (Borrelia_P83 family) basic membrane protein, putative Hemolysin

N-terminal signal peptide yes (all LP) yes (all LP) yes (1 LP) yes (1 LP) yes yes (LP) yes (LP) yes yes no

OMV enrichment Ratios 0.04, 0.05, 0.06, 0.09, 0.10, 0.18 0.11, 0.28

Combined p-Value

0.02, 0.07 0.28, 0.28, 0.41 0.21, 0.33 0.04 0.09 0.10 0.13 0.17

0.0007 0.009

0.0006 0.013

0.002 0.001 0.009 0.001 0.018 0.003



Page 46 of 51

Table 3. Lipoproteins localised to the OM Locus

Description

MW (kDa)

Relative Abunda

Mascot Scoreb

Pfam Name

OMV Enrich c

TP Homologd

TDE0015

putative OM lipoprotein

37.6

6.3

504

0.89

TDE0048

24.3

2.1

342

2.35

11.4

3.9

200

6.55

-

34.8 92.0 23.0

2.6 5.0 1.5

817 2471 403

3.69 6.15 1.28

-

31.5

0.17

2.09

-

39.9

1.8

2.66

-

0.94

-

3.88 2.58 1.92 8.56 3.43 8.19 1.23

-

3.82

-

10.61

-

TDE0939

putative OM lipoprotein Factor H-like 1 binding protein, FhbB putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein peptidase, M48 family lipoprotein TPR domain protein sialidase domain lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein dentilisin associated protein , PrcB dentilisin associated protein , PrcA dentilisin protease putative OM lipoprotein putative OM lipoprotein phosphatase/nucleotidase lipoprotein putative OM lipoprotein

TP0134 TP0133 -

TDE0940

putative OM lipoprotein

TDE0108 TDE0117 TDE0139 TDE0324 TDE0410 TDE0430 TDE0471 TDE0571 TDE0572 TDE0649 TDE0730 TDE0731 TDE0732 TDE0753 TDE0760 TDE0761 TDE0762 TDE0803 TDE0833 TDE0870

TDE0941 TDE0993 TDE1190 TDE1232 TDE1246 TDE1498 TDE1557 TDE1642 TDE1660 TDE1663 TDE1693 TDE1940 TDE2023 TDE2055

NLP/P60 family lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein Putative YfiO/BamD lipoprotein putative OM lipoprotein putative OM lipoprotein Leucine Rich Repeat domain lipoprotein OmpA family lipoprotein putative OM lipoprotein putative OM lipoprotein TPR domain lipoprotein OM hemin-binding protein B (HbpB)

138 Peptidase_M48 1157 TPR_1 Sialidase nonvirale FGE-sulfatase FGE-sulfatase

59.8

0.46

257

83.1 41.2 12.7 52.5 24.5 48.9 24.7

0.72 1.6 1.9 2.3 84 7.9 1.7

616 955 324 1512 1540 1376 385

17.6

0.81

69.7

23

77.5 56.0 37.3

14 3.5 0.74

2596 Peptidase_S8 1387 376 FGE-sulfatase

6.40 1.77 1.78

-

71.5

0.24

386 5_nucleotid_C

1.82

TP0104

35.4

2.4

525 TraB

4.26

16.0

0.88

188

2.56

TP0675 WP_01434 2851.1

17.9

0.54

177 NLPC_P60

3.23

-

54.8 16.6 76.4 89.7

0.48 20 0.10 0.19

945 TPR_19 919 170 FGE-sulfatase 952 Chlam_PMP

1.06 4.25 0.48 0.66

TP0954 -

55.2

0.01

83 YfiO/BamD

0.57

TP0369

26.6 26.9

0.48 10

1.93 1.92

TP0772

164.4

0.05

188 LRR_5

0.95

TP0225

19.2 29.1 40.6 43.5

1.3 7.8 0.99 0.03

307 OmpA 1109 360 43 TPR_11

0.41 6.44 1.20 0.84

TP0471

44.8

18

2042 ZinT

1.06

-

307 PrcB_C 4502

316 1276


Page 47 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TDE2056 TDE2068 TDE2101 TDE2104 TDE2147 TDE2164 TDE2211 TDE2242 TDE2257 TDE2258 TDE2269 TDE2350 TDE2419 TDE2495 TDE2515 TDE2591 TDE2662 TDE2693 TDE2714 TDE2735 TDE2737


OM hemin-binding protein A (HbpA) putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein Putative YfiO/BamD lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein 5-nucleotidase family lipoprotein surface antigen BspA, putative lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein putative OM lipoprotein rhodanese-like domain lipoprotein putative OM lipoprotein ankyrin repeat lipoprotein putative OM lipoprotein surface antigen, putative lipoprotein putative OM lipoprotein

45.2

57

90.9 39.0 94.7

0.04 0.18 0.21

17.5

2873 ZinT

1.14

-

100 499 FGE-sulfatase 165

1.04 0.77 0.79

-

3.5

398 YfiO/BamDe

0.97

TP0625

25.8 22.0 90.9

1.6 1.6 0.12

474 371 222

1.01 1.62 1.10

-

57.8

9.1

1.04

TP0104

37.2

2.8

473 LRR_5

3.62

TP0225

35.6 23.1 26.0 83.7 38.9

1.4 4.7 1.6 0.08 0.98

430 649 625 57 868

3.88 2.51 2.09 3.14 6.10

TP0503 -

13.7

3.6

347 Rhodanese

0.65

-

32.4 102.0 74.0

0.49 0.06 0.97

259 495 Ank_2 518 FGE-sulfatase

1.70 0.43 5.63

TP0453 TP0835 -

49.1

47

3730 LRR_5

5.62

-

26.6

17

1078 LRR_5

3.36

1770 5_nucleotid_C

FGE-sulfatase DUF2807 FGE-sulfatase FGE-sulfatase

a

The average relative abundance in the OMV replicates. This value is based on the iBAQ values with the most abundant OMV protein being set to 100 (See Table S2). b The best Mascot score obtained from the OMV replicates (see Table S1). c The OMV enrichment ratio was calculated as iBAQ (OMV) / iBAQ (Cells). d Orthologs found in T. pallidum by BLAST search are provided. e From NCBI Conserved Domain Search (not Pfam).



Page 48 of 51

Table 4. OM β-barrel proteins identified in OMVs. Locus TDE0014 TDE0308 TDE0405 TDE0467 TDE0688 TDE0981 TDE1231 TDE1234 TDE1327 TDE1489 TDE1490 TDE1554 TDE1666 TDE1717 TDE1848 TDE1884 TDE2011 TDE2117 TDE2285 TDE2308 TDE2508 TDE2567 TDE2601 TDE2673 TDE2674

Description predicted βbarrel Omp predicted βbarrel Omp major outer sheath protein predicted βbarrel Omp predicted BamA-like protein predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp OM efflux protein, putative predicted βbarrel Omp predicted βbarrel Omp predicted βbarrel Omp β-barrel assembly protein, BamA predicted βbarrel Omp predicted βbarrel Omp

MW (kDa)

Relative Abunda

Mascot Scoreb

OMV Enrichc

TMBB Scored

Structural Homologe OmpA, 98%, 75% NanC porin 70%, 55%

TP Homologf

22.9

2.1

139

0.62

0.998

TP_0733

40.2

0.04

153

0.98

0.996

58.3

37

3888

0.68

1

53.2

0.41

313

1.12

0.912

Momp porin, 98%, 90%

-

58.4

0.08

116

0.34

1

BamA, 98%, 91%

-

36.2

2.5

574

1.79

0.978

64.7

1.7

756

1.53

0.686

63.6

0.52

835

6.30

0.466

43.1

1.3

703

2.44

0.969

-

30.8

8.1

335

8.36

0.901

-

43.7

1.1

563

2.72

0.94

-

39.5

0.01

34

0.09

0.96

-

27.1

0.05

40

3.14

0.613

-

23.2

39

949

1.45

0.92

53.8

2.5

707

1.07

0.997

36.8

4.4

843

12.90

0.972

54.8

0.01

127

1.44

0.992

TP_0897

Wzi, 100%, 70% cov Wzi, 98%, 74%

OmpW, 82%, 73% Alg exp prot, 95%, 53% TbuX, 100%, 95% PhoE porin, 99%, 87%

-

TP_0856 TP_0548 TP_0859 TP_0865 TP_0966

47.7

0.19

285

2.98

0.995

TbuX, 100%, 70%

53.5

0.06

67

0.25

0.978

TolC, 99%, 93%

22.5

7.7

449

0.55

0.991

-

50.7

25

1461

0.92

0.998

-

32.3

8.2

544

2.51

0.976

-

93.8

3.3

1676

0.62

0.997

25.5

100

2141

2.88

0.431

29.4

7.9

740

11.30

0.656

BamA, 100%, 95% OprG, 92%, 60% OmpW, 82%, 50%

TP_0326 -

a

The average relative abundance in the OMV replicates. This value is based on the iBAQ values with the most abundant OMV protein being set to 100 (See Table S2). b The best Mascot score obtained from the OMV replicates (see Table S1). 48 ACS Paragon Plus Environment

Page 49 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


c The

OMV enrichment ratio was calculated as iBAQ (OMV) / iBAQ (Cells). TMBB score predicts the likelihood of the protein adopting a trans-membrane β-barrel fold. e Structural homolog found by Phyre 2, together with the score and sequence coverage. f Orthologs found in T. pallidum by BLAST search are provided. d The



Page 50 of 51

Table 5. MS identification of proteins from T. denticola OMVs separated by 2D-BN-PAGE Spota

Locusb

A B C D

NR TDE0139 TDE0762 TDE0761

E

TDE0761

F

TDE0985

G

TDE1071

H

TDE1273

I

TDE2735 TDE2257 TDE0398

J

TDE0386

K

TDE0951 TDE2226

L M N1

TDE0405 TDE2508 TDE2056 TDE2055 TDE2056 TDE2055 TDE0929 TDE0902 TDE0981 TDE1693 TDE2737 TDE2673 TDE1717

N2 O P

Q R S T U V W

TDE1234 TDE2735 TDE2369 TDE2217

X Y

TDE1950 TDE2258

Z

TDE2567

Name/description

Mascot Ions score

# Peptides

Obs.c Native Ma (kDa)

putative OM lipoprotein dentilisin protease dentilisin associated protein, PrcA2 (C-terminal fragment of PrcA) dentilisin associated protein, PrcA1 (N-terminal fragment of PrcA) oligopeptide/dipeptide ABC transporter, periplasmic peptidebinding protein peptide ABC transporter, peptidebinding protein OppA oligopeptide/dipeptide ABC transporter, peptide-binding protein surface antigen, putative lipoprotein 5-nucleotidase family lipoprotein oligopeptide/dipeptide ABC transporter, periplasmic peptidebinding protein ABC transporter, periplasmic substrate-binding protein Putative IM lipoprotein ABC transporter, substrate-binding protein, putative major outer sheath protein predicted β-barrel Omp OM hemin-binding protein A (HbpA) OM hemin-binding protein B (HbpB) OM hemin-binding protein A (HbpA) OM hemin-binding protein B (HbpB) ornithine carbamoyltransferase (argF) putative OM-associated protein predicted β-barrel Omp putative OM lipoprotein putative OM lipoprotein predicted β-barrel Omp predicted β-barrel Omp serum albumin predicted β-barrel Omp surface antigen, putative lipoprotein conserved domain protein galactose/glucose-binding lipoprotein (mglB) putative IM lipoprotein, TmpC surface antigen BspA, putative lipoprotein predicted β-barrel Omp

187 471 232

9 8 7

310 370-480 370-480

93/92 70/78 38/40

276

7

370-480

34/30

248

12

180

74/75

202

8

200

66/67

393

8

190

54/60

528 506 379

14 12 16

260-350

54/50 54/58 54/59

715

18

510

37/37

403 221

12 6

220-290

36/38 36/37

680 638 884 586 617 395 180 234 257 263 222 809 151 487 544 506 477 214

10 13 21 16 15 12 8 8 8 7 8 7 4 14 16 10 13 7

480-580 650-720 260-370

120-270 240 60 240-300 270-340 180-320 150

52/58 48/51 43/45 43/45 43/45 43/45 38/38 28/27 28/36 28/29 28/27 23/25 17/23 70/71 57/64 49/50 42/41 40/43

305 171

8 4

180-320 80

38/39 34/38

189

5

50-100

30/32

430-650 600 260-320

Obs./Calc.d 2nd-Dim MW (kDa)

a Identified

spot in Figure 5. no matching result from MS analysis. c Observed Native molecular mass (M ) for spot was estimated from 2D-BN-PAGE gel in Figure 5. a d Observed second-dimension MW for spot was estimated from 2D-BN-PAGE gel in Figure 5 Calculated MW for proteins identified were from full-length amino acid sequences. b NR,


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