Protein interactions between the oral bacteria Fusobacterium nucleatum and Porphyromonas gingivalis in biofilm and planktonic culture

The opportunistic pathogens Fusobacterium nucleatum and Porphyromonas gingivalis are Gram-negative bacteria associated with oral biofilm and periodontal disease. Although liquid cultures are often the preferred cultivation method in microbiology, bacterial cells in biofilm adopt a profoundly different phenotype reflecting the close cell-to-cell contact compare to their planktonic counterparts. To investigate F. nucleatum and P. gingivalis interactions relevant in biofilm formation, we applied liquid chromatography-tandem mass spectrometry to determine the expressed proteome of F. nucleatum and P. gingivalis cells that were grown either as biofilm or in planktonic culture, and individually or together. The proteomic analyses detected 1,322 F. nucleatum and 966 P. gingivalis proteins. We statistically compared the proteins label-free quantitative (LFQ) intensities between biofilm and planktonic culture and identified significant changes (p-value ≤0.05) in 0,4% F. nucleatum proteins, 7% P. gingivalis proteins, and more than 14% of all proteins in the dual-species model. For both species, proteins involved in vitamin B2 (riboflavin) metabolic process had significantly increased levels in the biofilm condition. In both mono- and dual-species biofilm models, P. gingivalis increased the production of proteins functional in translation, oxidation-reduction, and amino acid metabolism, when compared to planktonic cultures. However, when we compared LFQ intensities between mono- and dual-species models, over 90% of the significantly changed P. gingivalis proteins had their levels reduced in biofilm and planktonic settings of the dual-species model. Our findings suggest that the two bacteria interact with each other at the protein level and indicate that P. gingivalis reduces the production of multiple proteins because of more favourable growth conditions provided by F. nucleatum presence. The results highlight the complex interactions of bacteria contributing to oral biofilm, which need to be considered in the design of future prevention strategies.


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Fusobacterium nucleatum and Porphyromonas gingivalis are important colonizers of the 48 subgingival biofilms (1). Both bacteria play a role in the pathogenesis of periodontal diseases, 49 a group of inflammatory diseases of the teeth supporting tissues (2). A mild form called 50 gingivitis is highly prevalent and can affect up to 90% of the worldwide population. However, 51 gingivitis does not affect the underlying supporting structures of the teeth and is reversible. A 52 more severe form of the disease, periodontitis, results in loss of connective tissue and bone 53 support and is the main cause of tooth loss in adults (3).

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F. nucleatum is commonly cultivated from the subgingival biofilm and tends to aggregate 55 with other oral bacteria, working as a bridge between early and late colonizers in the 56 development of the dental biofilm (4). P. gingivalis is a member of the Socransky's red 57 complex, a group of bacteria strongly associated with periodontal disease (5). It is often found 58 in the deep periodontal pockets, and it produces a broad array of potential virulence factors 59 involved in tissue colonization and destruction as well as in perturbations of the host defence 60 (reviewed in (6-8)). 61 P. gingivalis and F. nucleatum are considered strict anaerobes, and both species display a 62 synergistic enhancement in biofilm formation and pathogenicity (9, 10). Several studies 63 showed that the bacteria could grow in a partially oxygenated condition when grown together 64 and suggested that enhanced production of oxidoreductive enzymes by F. nucleatum is 65 protecting P. gingivalis from oxidative stress (11,12). Similarly, in vitro and in vivo models 66 showed a nearby association between the bacteria, indicating that they co-aggregate and 67 potentially support each other (10,13,14). However, how the bacteria interact at the protein 68 level remains poorly understood.
Although most microbiology research has been focused on free-floating bacteria in 70 suspension (planktonic cells), mounting evidence indicates that cells growing in biofilms are 71 in a very different physiological state (15)(16)(17). For example, the envelope fraction of 72 Pseudomonas aeruginosa cells grown as a biofilm showed a 30-40% difference in the 73 detected proteins compared with the same fraction of P. aeruginosa planktonic cells (15). 74 Recent investigations indicate that biofilm is the preferred form of life for most microbes, 75 particularly those of pathogenic nature (18). 76 In the current study, we explored the differences between cells grown in a culture or biofilm 77 at the proteome level. To address the synergistic relationship between F. nucleatum and P. 78 gingivalis, we grew the bacteria both individually and in a dual-species model. The results 79 suggest that the two bacteria actively interact with each, and P. gingivalis reduces the 80 production of certain proteins in the biofilm, possibly because of a favourable presence of F. 81 nucleatum proteins that support the necessary biochemical adaptations. Trasadingen, Switzerland). The flasks were incubated at 37°C for four days. The medium was 95 then removed and the biofilm samples were washed once with phosphate buffered saline 96 (PBS), before the biofilms were harvested with cell scraper (Nunc, Rochester, NY, USA). The 97 biofilm samples were resuspended in 500 µl PBS and stored at -20°C until further processing.

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The planktonic cultures were anaerobically grown in the same liquid medium as described 99 above in 10 ml glass round bottom test tubes with screw caps at 37°C, without shaking. After 100 four days, the bacteria were collected by centrifugation at 3000 x g for 3 min in room 101 temperature. The pelleted cells were resuspended in 500 µl PBS and stored at -20°C until 102 further processing.

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Protein extraction from the biofilm and planktonic samples 104 The bacterial cells were washed 3 times by resuspension in 1 ml PBS and centrifugation for 105 10 min each time at 6000 x g at +4°C. In a final step, the cells were resuspended in 1 ml of extraction buffer (10 mM Tris-HCl, 2.5 % SDS, pH 8.0). The cell suspensions were 107 transferred to FastPrep® Lysing Matrix A, 2 mL Tube (MP Biomedicals, California, USA) 108 and then bead-beated in FastPrep® FP120 Cell Homogenizer (Thermo, California, USA) for 109 45 sec at 6.5 m/s. The cell extracts were cooled on ice for 5 min, then the cell debris was  Filtration and desalting 132 StageTips to be used for filtration and peptide samples desalting were prepared in-house 133 according to the protocol developed by Rappsilber and colleagues (20). Shortly, 3M Empore 134 C18 extraction disks (3M, Minnesota, USA) were packed in 200 µl pipet tips by a blunt-135 ended needle and a plunger or metal rod that helped fit the extracted disks in the pipet tips.

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The disks were then wetted by passing 20 µl of methanol, followed by 20 µl of elution buffer  identified by site and reverse hits. Each protein identified in at least two of the three replicates 185 was considered valid. Proteins with significant differential levels were identified by statistical 186 analysis based on two-sided t-test, which was performed on proteins log 2 transformed LFQ 187 values. A protein was considered significantly changed if it was marked as significant in the t-188 test and showed more than 2 log 2 difference form the the mean LFQ intensity.

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This study's objective was to investigate F. nucleatum and P. gingivalis proteins relevant in 198 biofilm formation. We grew the bacteria in mono-or dual-species model either as biofilms or  Table 1. 209 The proteome coverage, which we defined as the number of the detected proteins divided by 210 the theoretical proteome derived from the UniProt database, was higher for F. nucleatum (≈ 211 62%) than the one of P. gingivalis (≈ 43%), both in biofilm and planktonic conditions ( LFQ intensities covered a dynamic range of ≈ 12 log 2 (Fig S1, Supplementary file 2), and the 224 correlations between replicates, represented as the Pearson correlation coefficient, varied 225 between 0.79-0.98 (Fig S2, Supplementary file 2).

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The most abundant proteins identified in the biofilm ( Five of ten most abundant F. nucleatum and P. gingivalis proteins detected in the planktonic 243 culture were also identified as the most abundant in the biofilm (Table 2 and 3). Yet, none of 244 these proteins were among significantly changed proteins (see below) between the planktonic

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To identify proteins produced in differential amounts by cells either in the biofilm or in 259 planktonic culture, we statistically compared LFQ intensity readings by the student t-test 260 (p≤0.05). Similarly, we compared the LFQ intensities of proteins produced by cells grown 261 under either mono-or dual-species conditions ( Table 4).

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* Proteins that passed the t-test and showed more than 2 log 2 difference in the LFQ intensity. planktonic culture, showed a significant change in their LFQ levels (Fig 1A). Proteins with 274 significantly increased levels in the biofilm (Table S4)

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Some of the proteins associated with F. nucleatum pathogenicity were identified as 284 significantly different (p ≤0.05) but showed less than 2log 2 difference between the biofilm 285 and planktonic cultures (Table S4). For example, metal-dependent hydrolase (FN1210), a 286 resistance causing and drug efflux protein with beta-lactamase activity (38), had slightly 287 increased LFQ levels in the biofilm mode of growth. Three other proteins (FN1613, FN0268, had also increased levels in the biofilm.

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to the planktonic condition 292 Approximately 7% of all P. gingivalis proteins quantified under biofilm and planktonic 293 culture (40 out of 593) showed significant changes in their LFQ levels ( Table S5). We 294 detected 30 proteins with more than 2log 2 LFQ levels increase in the biofilm setting (Fig 1B). 295 As in F. nucleatum biofilm, riboflavin biosynthesis protein (RibBA) had increased levels in 296 the P. gingivalis biofilm. The latter coincides with a transcriptomic study, which showed that 297 the protein is upregulated in a P. gingivalis biofilm (39). This protein is involved in biofilm-

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We identified ten P. gingivalis proteins with increased amounts in the planktonic condition, 303 including outer membrane efflux protein (PGN_1432) that has cellular transport activity, an 304 integral component of membrane (PGN_0296), immunoreactive 23 kDa antigen protein 305 (PGN_0482), and membrane-associated zinc metalloprotease (PGN_1582) that has peptidase 306 activity and is involved in proteolysis. These results support findings from a gene expression 307 analysis study, which showed that P. gingivalis genes are upregulated in the biofilm setting 308 compared to planktonic culture (39).

A majority of P. gingivalis proteins have increased levels in biofilm compared to planktonic
310 culture when cultured as dual-species model with F. nucleatum. 311 In the mixed-species cultures, 797 proteins were quantified under both biofilm and planktonic 312 conditions, and LFQ intensities of 14% (112) proteins were significantly changed (Fig 1C). 313 Among these proteins, we detected more proteins derived from F. nucleatum (72) than P. 314 gingivalis (40). Of 78 proteins with increased amounts in the biofilm (Table S6)  Eleven out of 635 F. nucleatum proteins that were quantified both in the mono-and dual-340 species biofilm showed significantly different levels (Fig 2A and Table S7). The three  Table S8). Most of these proteins were annotated as uncharacterized. Thus, the 349 results suggest that F. nucleatum responds to the presence of P. gingivalis only mildly, mostly 350 by decreasing production of specific proteins. 352 We identified 283 P. gingivalis proteins that were quantified both in the mono-and dual-353 species biofilms, and most of the proteins with significantly different levels were decreased in 354 the dual-species biofilm (51 out of 56) (Fig 2C and Table S9). Functional analysis of these 355 proteins pointed out the following functional clusters: structural ribosomal activity (6 356 proteins), translation (6 proteins), oxidation-reduction (4 proteins), and RNA binding (4 357 proteins). The reduction in amounts of multiple proteins agreed with a proteomic study of P. provided physiologic support to P. gingivalis and, in this way, reduced its stress.

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Statistical comparison of P. gingivalis proteins LFQ intensities between the mono-species and 362 dual-species planktonic cultures identified 63 significantly different proteins, and 59 of these 363 showed reduced levels in the dual-species culture (Fig 2D and