Proteomic Analysis of the Periodontal Pathogen Prevotella Intermedia Secretomes in Biofilm and Planktonic Lifestyles

Proteomic Analysis of the Periodontal Pathogen Prevotella Intermedia Secretomes in Biofilm and Planktonic Lifestyles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Proteomic Analysis of the Periodontal Pathogen Prevotella Intermedia Secretomes in Biofilm and Planktonic Lifestyles Maribasappa Karched, Radhika Bhardwaj, Muawia Qudeimat, Areej Al-Khabbaz, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1078670/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Apr, 2022 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Prevotella intermedia is an important species associated with periodontitis. Despite the remarkable clinical significance, little is known about the molecular basis for its virulence. The aim of this study was to characterize the secretome of P. intermedia in biofilm and planktonic life mode. The biofilm secretome showed 109 proteins while the planktonic secretome showed 136 proteins. The biofilm and the planktonic secretomes contained 17 and 33 signal-peptide bearing proteins, 13 and 18 lipoproteins, respectively. Superoxide reductase, sensor histidine kinase, C40 family peptidase, elongation factor Tu, threonine synthase etc. were unique to biofilm. Of the ~30 proteins with predicted virulence potential from biofilm and planktonic secretomes, only 6 were common between the two groups, implying large differences between biofilm and planktonic modes of P. intermedia . From gene ontology biofilm secretome displayed a markedly higher percent proteins compared to planktonic secretome in terms of cellular amino acid metabolic process, nitrogen compound metabolic process etc. Inflammatory cytokine profile analysis revealed that only the biofilm secretome, not the planktonic one, induced important cytokines such as MIP-1a/MIP-1b, IL-1b, and IL-8. In conclusion, the revealed differences in the protein profiles of P. intermedia biofilm and planktonic secretomes may trigger further questions about molecular mechanisms how this species exerts its virulence potential in the oral cavity. General Microbiology Periodontal Pathogen Planktonic Secretomes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Bacteria release proteins and other components into extracellular milieu continuously during normal growth and physiology. Many of the secreted proteins often function in nutrient acquisition, but in pathogenic bacteria, they play a key role in disease, e.g., by helping the bacteria in host colonization or by modulating host immune responses 1 , 2 . Thus, extracellular secretion of bacterial proteins is an important virulence mechanism. To achieve this, bacteria have devised various strategies, e.g., dedicated secretory systems 3 , 4 and extracellular vesicles 5 , 6 . The central components of the main protein translocation system, the Sec system, share a high degree of sequence similarity between Gram-positive and Gram-negative bacteria. Oral infectious diseases such as caries and periodontitis are dental plaque biofilm-driven 7 , 8 . Dental plaque is a structurally and functionally organized, highly complex multispecies biofilm. The resident bacteria in this multispecies community exhibit extensive interactions while forming bioଁlm structures, carrying out physiological functions, and inducing microbial pathogenesis 9 . Interspecies interactions in biofilms are competitive, cooperative and, antagonistic 10 . To facilitate such interactions, plaque bacteria may release several cellular components into the extracellular space within the biofilm matrix. In addition, proteins secreted in biofilms may have specific effects in terms of the virulence properties of bioଁlm residents, which could inଂuence the overall pathogenicity of biofilms. Periodontitis is a chronic inflammatory disease characterized by mild to moderately severe inflammation of the periodontal tissue, progressive destruction of ligament fibers, as well as alveolar bone loss 11 . The disease is primarily related to chronic plaque accumulation in a susceptible host. Major bacterial species implicated in periodontitis are, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans and others 8 , 12 . Prevotella intermedia occurs frequently in the subgingival samples of periodontitis patients 8 . Further, presence of Prevotella has been positively correlated with clinical attachment loss 13 , bleeding on probing 14 , and periodontal inflammation 15 . P. intermedia is a Gram-negative, non-motile, rod-shaped, bacterium that requires strict anaerobic conditions for growth. In addition to oral infections, P. intermedia has also been detected from nonoral sites, e.g., NOMA (cancrum oris) lesions 16 and bacterial tracheitis in children 17 . Further, chronic oral infections such as periodontitis in which P. intermedia is a major species, increase the risk of systemic diseases, such as atherosclerosis, pre-term delivery of low birth-weight infants 18 , 19 . Importantly, P. intermedia is known to be resistant to several antibiotics including cephalosporins, penicillins and tetracyclins 20 – 22 . Since P. intermedia is not an exogenous pathogen and is a part of normal oral microbiota, its complete elimination is not possible. Despite the remarkable clinical significance P. intermedia has, little is known about the molecular basis for its virulence. For P. intermedia to survive in a complex and competitive oral environment, it is imperative that it can adhere to surfaces and integrate into plaque biofilm. In general, bacterial cells dispersed from mature plaque biofilms are collected by saliva and can be regarded as planktonic cells. Such planktonic bacterial cells can reattach to oral surfaces and initiate new biofilm growth. Recent knowledge from the literature suggests that virulence potentials of the biofilms and the planktonic cells are remarkably different 23 . Previously, even though different aspects of P. intermedia as part of plaque biofilm have been studied, extracellular proteins secreted by this species (secretome), in biofilm or in planktonic life form, has not been investigated. Results Analysis of the secretomes of P. intermedia biofilm and planktonic cells . Protein preparations (Fig. 1) from P. intermedia were analyzed by LC-MS/MS. Database search (NCBI-nr) revealed 109 proteins from the biofilm (Suppl. File S1) and 136 proteins from planktonic cells (Suppl. File S2). To ensure that the secretome preparations from the biofilms and the planktonic cells did not contain cytoplasmic proteins due to cell lysis, western blot analysis was performed using an antibody against a cytoplasmic marker protein FtsZ. Panel B in Fig 1 shows the presence of the marker protein from the whole cell protein preparation from P. intermedia , but not from the secretome preparations. As depicted in a theoretical 2DE map of the secretome, the MW of the secreted proteins ranged between 5 kDa and 130 kDa (Fig. 1C). In both biofilm and planktonic cells, with respect to predicted pI values, majority of the proteins formed a cluster with the pI range of 4.0 and 6.5. Protein sequences of the secretomes of P. intermedia biofilms and planktonic cells were analyzed for the route of their extracellular release by various bioinformatics tools. Biofilm and planktonic preparations showed 17 and 33 signal peptide-bearing, 13 and 18 lipobox bearing and 2 and 0 TatP signal bearing proteins respectively. Transmembrane alpha helices were found in 4 and 6 proteins from biofilm and planktonic preparations. Subcellular localization analysis (Fig. 2) revealed that the secretome from planktonic cells contained more extracellular proteins (11.6%) than the biofilms (7.4%). Proteins of cytoplasmic origin were more in the biofilm (56.4%) than the planktonic cells (50%). Further, 40-50% of the proteins identified from the secretomes were unique to biofilm or planktonic cells. Importantly, proteins such as superoxide reductase, sensor histidine kinase, C40 family peptidase, elongation factor Tu, threonine synthase etc. were unique to biofilm secretome and were not detected in the planktonic secretome. Potential virulence proteins in P. intermedia secretome. Virulence potential of the P. intermedia was assessed by in silico prediction of virulence factors using the online tools “VirulentPred” and “VFDB” (Virulence Factor DataBase). We found that 31 proteins from the biofilm secretome and 30 proteins from planktonic secretome were predicted to be virulent (Tables 1 and 2). Hemin-binding protein, porin family protein, OmpA, thioredoxin, Omp-28, tetratricopeptide-binding protein molecular chaperones DnaK and GroES, were the major proteins with demonstrated virulence properties in other bacterial species. Table 1. Proteins with predicted virulence properties from P. intermedia biofilm. Reference Number Protein WP_014710387.1 DNA starvation/stationary phase protection protein WP_028905462.1 Peptidase M6 WP_028905527.1 Sensor histidine kinase WP_028905084.1 gb|APW32430.1| hypothetical protein BWX39_07160 [Prevotella intermedia ATCC 25611 = DSM 20706]">Tetratricopeptide repeat protein WP_028905748.1 hemin-binding protein WP_014710403.1 thiol reductase thioredoxin WP_004356500.1 Xaa-Pro aminopeptidase WP_004364886.1 trypsin WP_028905224.1 peptidyl-prolyl cis-trans isomerase WP_013265509.1 NADH oxidase WP_028904949.1 peptide ABC transporter substrate-binding protein WP_018667829.1 ATPase WP_015531502.1 MFS transporter WP_172460529.1 ROK family protein WP_028904772.1 urocanate hydratase WP_028904668.1 ribulose-phosphate 3-epimerase WP_007835729.1 Asp/Glu/hydantoin racemase WP_005332057.1 N-acetylmuramoyl-L-alanine amidase WP_007133390.1 Molecular chaperone DnaJ MBP5257375.1 acetyltransferase WP_028906355.1 gb|APW31683.1| AraC family transcriptional regulator [Prevotella intermedia ATCC 25611 = DSM 20706]">substrate-binding domain-containing protein WP_028904901.1 energy transducer TonB WP_007411110.1 anthranilate phosphoribosyltransferase WP_028906371.1 1-acyl-sn-glycerol-3-phosphate acyltransferase MBO5313912.1 Membrane protein M15 WP_028905881.1 multidrug ABC transporter ATP-binding protein MBF1618150.1 type IV secretion protein Rhs WP_028906306.1 L-asparaginase WP_014709317.1 YkgB family protein MBQ7451373.1 threonine synthase MBP3838531.1 HAD-IA family hydrolase Table 2. Proteins with predicted virulence properties from P. intermedia planktonic cells. Reference Number Protein WP_014710403.1| thiol reductase thioredoxin WP_028905059.1| trypsin WP_025000944.1| DNA starvation/stationary phase protection protein WP_014709654.1| peptidase M6 WP_028906361.1| DNA topoisomerase II WP_028905748.1| hemin-binding protein WP_028905169.1| DUF4595 domain-containing protein WP_014709619.1| enoyl-ACP reductase WP_028905189.1| PorT family protein MBR7087708.1 amino acid adenylation domain-containing protein MBP7359878.1 chemotaxis protein MBO7539992.1 DNA polymerase III subunit gamma/tau MBQ3767790.1 ATP-binding protein WP_014709212.1| peptidylprolyl isomerase MBO7578384.1 TolC family protein WP_014709366.1 ABC transporter MBP8758149.1 DUF1622 domain-containing protein MBR2882634.1 ankyrin repeat domain-containing protein WP_099836288.1 DNA-binding response regulator WP_097549978.1 gb|PDP60535.1| tRNA epoxyqueuosine(34) reductase QueG [Prevotella intermedia]">tRNA epoxyqueuosine(34) reductase QueG MBA7488061.1 calcineurin-like phosphoesterase C-terminal domain-containing protein MBP5424796.1 sigma-70 family RNA polymerase sigma factor WP_100190220.1 peptidase M20 WP_100356678.1 gb|PJI24114.1| type IV secretion protein Rhs [Prevotella intermedia]">type IV secretion protein Rhs WP_088437864.1 ABC transporter ATP-binding protein WP_099984831.1 glycosyltransferase MBP9983829.1 MarR family transcriptional regulator WP_099976545.1 gb|PIN28473.1| two-component sensor histidine kinase [Prevotella intermedia]">HAMP domain-containing histidine kinase RKW57308.1 SDR family oxidoreductase WP_097656281.1 TonB-dependent receptor Gene ontology analysis Gene Ontology (GO) analysis of the amino acid FASTA sequences of the P. intermedia secretomes was achieved by using the tools Blast2GO and CELLO2GO. As shown in Fig. 3, marked differences in the percentage of proteins was found with GO annotations in “biological processes” and “molecular functions”. In the category biological processes, biofilm secretome showed higher number of proteins in the case of catabolic process, pathogenesis, cellular amino acid metabolic process, small molecule metabolic process, cellular nitrogen compound metabolic process, while proteins in the “transport” group were higher in planktonic secretome. In the category “molecular function”, biofilm showed higher proteins with “protein binding”, “methyltransferase” and “kinase” activities. The planktonic secretome showed higher number of proteins with peptidase activity. Functional protein association network analysis As seen in Fig. 4, P. intermedia secretome proteins formed three major groups in the STRING network, i.e., carbohydrate metabolism, ribosomal proteins, and chaperones/virulence proteins. Components of the sugar metabolism network were glutamate dehydrogenase, glucose-6-phosphate isomerase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphate aldolase, serine hydroxymethyltransferase, and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase. Putative virulence-associated proteins and molecular chaperones such as thioredoxin, DnaK, dps, and GroEL formed another cluster. The ribosomal protein cluster included rplF, rpsE, rpsM, rpsB rSA2, and rplL5 in both biofilm and planktonic cells (Fig. 4). Inflammatory potential of the P. intermedia secretomes To get a preliminary insight into the inflammatory potential, human whole blood was stimulated with P. intermedia secretomes from biofilm and planktonic cultures. As determined by signal densities of cytokine spots on a membrane array (Fig. 5), secretome preparations from both biofilm and planktonic cells induced similar levels of CCL5/RANTES, ICAM-1, MIF, and Serpin E1. The biofilm secretome additionally induced cytokines MIP-1a/MIP-1b, IL-1b, and IL-8. Discussion Protein secretion is a fundamental physiological process among bacteria. However, the components of the secretome may vary depending on the niche where the bacteria are located. Biofilm life is a modus operandi for most oral bacteria. Bacterial residents of plaque biofilms continuously release cellular components into the extracellular environment in the biofilm. Interestingly, in this study, proteins such as superoxide reductase, sensor histidine kinase, C40 family peptidase, elongation factor Tu, threonine synthase, which are known to play important roles in bacterial virulence and colonization in the host, were unique to biofilm secretome. Thus, fundamental differences, including those with respect to protein secretion, may exist between the biofilm and planktonic mode of life. The possibility of contamination of the secretome preparations with subcellular proteins resulting from cell lysis was ruled out by using an established cytoplasmic marker protein Fts-Z 24 which was undetected in all P. intermedia secretome preparations. Potential virulence factors in the secretome preparations were predicted by in silico analysis. While the total number of predicted virulence factors did not differ between the two secretomes (biofilm=31, planktonic=30), only 6 proteins, including thioredoxin, trypsin and hemin-binding protein, were common between them. This is an interesting finding given the fundamental differences of bacteria in biofilm and planktonic modes of life. In the subgingival environment where P. intermedia is part of the plaque biofilm encountering a multitude of host challenges, it probably needs to secrete an entirely different set of proteins with virulence potential compared to the planktonic form of life. Of noteworthy virulence factors in the biofilm secretome were thioredoxin, hemin-binding protein, peptidase M6, NADH oxidase and trypsin. In the case of planktonic secretome, enoyl-ACP reductase, PorT family protein, peptidase M20, and glycosyltransferase were some of the important ones. Interestingly, histidine kinases, which were found in both biofilm and planktonic secretomes, are recently proposed to be attractive antibacterial drug targets 25 . Further, hemin-binding protein that plays a role in bacterial interaction with host cells 26 , glycosyltransferases that mediate biofilm formation 27 . Similarly, virulence potential of other proteins such as NADH oxidase 28 and thioredoxin 29 has been well established in several other bacterial pathogens. To get an insight into the functional significance of the proteins identified from the P. intermedia proteomes, gene ontology analysis was carried out on the fasta sequences. The biofilm secretome contained higher percentage of proteins in “biological processes” category with catabolic process, cellular amino acid metabolic process, cellular nitrogen compound process. Biofilm secretome also showed higher number of proteins with protein binding, methyl transferase and kinase activities. Interestingly, the planktonic secretome appeared to possess higher percentage of proteins with peptidase activity, compared to the biofilm secretome. In another important periodontal pathogen P. gingivalis , certain peptidases were important for the survival of the species but not sufficient for its virulence 30 . The biofilm and planktonic secretomes were analyzed for their functional network associations by using STRING. Three function-based groups were identified, carbohydrate metabolism, ribosomal proteins and a third network comprising of virulence factors and chaperones. Key enzymes in carbohydrate metabolism, glutamate dehydrogenase, glucose-6-phosphate isomerase and phosphoenolpyruvate carboxylase were part of the network. Predicted virulence factors that also function as molecular chaperones, such as thioredoxin, DnaK, dps and GroEL formed a distinct network. Several years ago, the ability of P. intermedia DnaK to function as a molecular chaperone was assessed by a luciferase folding assay 31 . In periodontitis, stresses such as temperature and pH increase, increased flow of gingival crevicular fluid, and oxidative stress resulting from phagocytic cells. In response to these stresses, P. intermedia may show increased expression of heat-shock proteins such as DnaK. This may contribute to enhanced virulence of the species as well as its survival in stress conditions. In a study that investigated the effect of oxidative stress on P. intermedia protein exopression 32 , several proteins from the above functional networks, including fructose-1, 6-bisphosphate aldolase, reductases, ribosomal proteins and molecular chaperones like thioredoxin, DnaK were upregulated when bacteria were exposed to molecular oxygen. The significance of the secretion of these proteins may possibly be related to the ability of P. intermedia to travel beyond the oral cavity and colonize distant body sites where the species must adapt to oxygenated tissues. P. intermedia has been considered a major periodontal pathogen because of its established role in initiation and development of periodontitis by inducing a variety of proinflammatory cytokines, proteases, and matrix metalloproteinases. Important cytokines such as IL-8, IL1-b, and macrophage inflammatory proteins were induced by the biofilm secretome. P. intermedia and its components have been shown to induce IL-8, IL1-b, TNF-a and MMPs 33,34 . The role of these and other proinflammatory cytokines such as MIP- a and MIP- b in periodontitis has been well established 35 . Importantly, certain bacteria-secreted proteins stimulate cytokines only in their secreted-form, not when they are within the bacterial cell 36 . This type of specificity may suggest possible roles of P. intermedia secretome proteins beyond the oral cavity. In conclusion, the current proteomics data on the biofilm- and planktonic- secretomes may provide new insights into virulence mechanisms of P. intermedia . For example, the role/potential of the predicted virulence factors in the secretomes may be studied by overexpressing the respective genes in a suitable system, and by constructing specific knockout mutants for further studies using in vitro and/or in vivo models. Methods Biofilm and planktonic cultures P. intermedia ATCC 25611 was grown on brucella blood agar containing 5% sheep blood in anaerobic atmosphere at 37 °C for 3 days. Biofilms and planktonic cultures were grown as described earlier with some modifications. 37 Bacterial colonies were harvested from agar plates with sterile disposable loops and suspended in brucella broth. The bacterial cells were washed at least once by suspending in brucella broth and then collected by centrifugation at 5000×g for 5 min. The washed bacterial cell pellet was resuspended in 1 ml brucella broth to make a stock suspension. A final bacterial suspension of OD 600 =1 was prepared after measuring the optical density of the stock suspension. Biofilms and broth cultures for planktonic bacterial growth were initiated by inoculating 24-well plates and microfuge tubes, respectively, containing 900 µl brucella broth with a 100-µl aliquot from an OD 600 =1 suspension of each species. Wells or tubes with only broth were considered as negative control. The plates/tubes were incubated in the same culture conditions as above for 24 h. At the end of incubation period, supernatant broth from biofilms was aspirated and the supernatants from planktonic cultures were collected by centrifugation. These supernatants were subjected to secretome preparation. Preparation of secretome The secretomes were prepared by extracting proteins using tri-chloroacetic acid (TCA) precipitation method as described previously with modifications 38,39 . TCA stock (100% w/v) was mixed with supernatant culture broth at 1:4 ratio and incubated for 30 min at -20 ° C. After centrifugation at 14,000 ×g for 20 min at 4 ° C, traces of acid in the pellet were removed by washing twice with 0.5 ml cold acetone, followed by complete air-drying in a fume hood. The samples were desalted by ultrafiltration through 3K Ultra-0.5 centrifugal filter devices (Amicon) at 14,000 ´g for 15 min at 4 ° C. After discarding the flow-through, concentrates in the columns were finally eluted from columns by centrifugation at 1000 ´g for 2 min at 4 ° C. Broth without bacteria was incubated in parallel and used as negative control. Whole cell protein preparation P. intermedia colonies harvested from BBA plates were washed in sterile PBS by centrifugation at 5000 ×g for 5 min. The pellet was resuspended in lysis buffer containing 1 mg/ml lysozyme and 1 mM phenyl methyl sulfonyl fluoride (PMSF) and incubated for 4 h at 4 - 8 ° C. The samples were subject to sonication in Omni Ruptor at a pulse rate 40 for 8 times (1 min sonication with 1 min interval on ice). The lysates were centrifuged at 10,000 ×g for 10 min at 4 ° C. Determination of Protein Concentration Protein concentrations in secretome preparations and whole cell lysates were estimated by Quick Start TM Bradford protein microplate standard assay (Bio-Rad) as per manufacturer instructions. SDS-PAGE Protein samples were mixed with 5× Laemmli sample buffer (125 mM tris, pH 6.8; 6 % glycerol, 2 % SDS; 5 % beta-mercapthoethanol; 0.025 % bromophenol blue) and boiled at 95 °C for 5 minutes. The samples were loaded on a 15 % SDS-PAGE gel [4 % stacking gel (4 % acrylamide; 68 mM tris, pH 6.8; 0.2 % SDS), 15 % separating gel; 375 mM tris, pH 8.8; 0.1 % SDS]. Electrophoresis was run at 150 V for 75 minutes (Mini-protein II Dual Slab Cell, Bio Rad) and the protein bands were visualized using coommassie blue. Western blot analysis To ensure that the secretome preparations did not contain proteins that originated due to cell lysis, western blot analysis of whole cell lysate and secretome preparations was performed. Protein bands on the gel were transferred onto a PVDF membrane using Trans-Blot ® Turbo TM transfer system (Bio-Rad). To avoid unspecific binding, membrane was blocked with 5% skimmed milk overnight at 4 ° C. As primary antibody, an antibody against the cytoplasmic marker protein, Ftsz (Agrisera AB, Sweden) was used at 1:1000 dilution and incubated 1 h at room temperature. The membrane was then incubated as above with a peroxidase conjugated goat antirabbit secondary antibody (1: 5000). The membrane was washed with tris-buffer saline containing Tween-20 (TBST). Finally, the bound antibodies on the membrane were detected by using SuperSignal TM West Pico chemiluminescence substrate (Pierce) and images were acquired in G:Box Imaging System (Syngene). Identification of proteins by Nano-LC-ESI-MS/MS For the identification of proteins in the secretomes from biofilms and planktonic cultures, mass spectrometry was performed at Proteome Factory (Proteome Factory AG, Berlin, Germany) using nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With an Agilent 1100 nanoHPLC system (Agilent, Waldbronn, Germany) interfaced to an Orbitrap Velos (Thermo Scientific, Bremen, Germany) via a nanoelectrospray ion source. After pooling replicate samples from EVs preparations, proteins were reduced, alkylated and digested by trypsin (Promega, Mannheim, Germany). Then, 400 ng of the resulting peptides were subjected to the nanoLC-ESI-MS/MS. 1% acetonitrile/0.5% formic acid was used as eluent for 5 minutes to trap and desalt the peptides on the enrichment column (Zorbax 300SB-C18, 0.3 ´ 5 mm, Agilent). A water/acetonitrile (both supplemented with 0.1% formic acid) gradient from 5% to 40% acetonitrile was then used within 120 minutes to separate the peptides on a Zorbax 300SB-C18, 75 µm x 150 mm column (Agilent). The mass spectrometer automatically recorded mass spectra, and tandem mass spectra were data-dependently acquired for multiply charged ions. Protein identification was made using the Mascot search engine (Matrix Science, London, England) against the bacterial subset of the RefSeq protein database (National Center for Biotechnology Information), (downloaded on 1 st July 2016, 49867978 entries, NCBI, Bethesda, USA) and a database with common protein contaminants. For MS/MS spectra where assignment of the precursor ion’s charge state was missing, search parameters for ions from ESI-MS/MS data acquisition was set to "2+, 3+ or 4+" according to the instrument's and method's standard charge state distribution. The search parameters were: Fixed modifications: Carbamidomethyl (C); variable modifications: Deamidated (NQ), Oxidation (M); Peptide Mass Tolerance: ± 3 ppm; Fragment Mass Tolerance: ± 0.6 Da; Missed Cleavages: 2. The inclusion criterion was: peptides that match with a score of 20 or above. Mass spectrometry data, with the project acession number PXD029419, has been deposited at PRIDE archive ( https://www.ebi.ac.uk/pride/archive/ ) repository. The data files can be accessed with the username [email protected] and the password gtYGw1hi . Bioinformatics analysis Most of the bioinformatics analyses were performed as described previously with some modifications 40 . Theoretical in silico (2-DE) image of the proteins was acquired by using the tool JVirGel, version 2.0 ( http://www.jvirgel.de/index.html ) 41 . The subcellular localization of the secretome proteins was predicted using the PSORTb tool, version 3.0.2 ( https://www.psort.org/psortb/ ) 42 . Signal-peptide bearing proteins were predicted by using the online tool SignalP, version 5.0 ( http://www.cbs.dtu.dk/services/SignalP/abstract.php ) 43 . Lipoproteins in the secretomes were predicted using the prediction tools LipoP ( http://www.cbs.dtu.dk/services/LipoP/ ) and PRED-LIPO ( http://bioinformatics.biol.uoa.gr/PRED-LIPO/input.jsp ) 44 . Further, the prediction tool TatP ( http://www.cbs.dtu.dk/services/TatP/ ), was used to predict proteins secreted via the Twin-arginine translocation pathway (Tat-pathway) 45 . Function prediction analysis Gene Ontology (GO) of the proteins was analyzed by using the amino acid FASTA sequences. For this, GO annotations were analyzed and plotted using the tools OmicsBox version 1.3.11 ( https://www.biobam.com/download-omicsbox/ ) 46 , and CELLO2GO 47 . Functional association networks within the secretome proteins were determined using the tool STRING ( https://string-db.org/ ) 48 . Minimum interaction scores were set at a strong confidence level of 0.7. Prediction of virulence factors in the EVs proteomes VirulentPred ( http://203.92.44.117/virulent/ ) was utilized to predict potent virulence factors in the secretomes 49 , along with the Virulence Factor Data Base (VFDB; http://www.mgc.ac.cn/VFs/ ). Stimulation of human whole blood with secretome protein preparations Whole blood collected from a healthy human volunteer was stimulated with P. intermedia biofilm and planktonic secretome preparations for 24 hours. In a 24-well plate, whole blood was added to the wells and stimulated with 20-µg of secretome protein preparations. After incubating at 37 °C and in 5% CO 2 in air for 24 hours, plasma was separated by centrifugation at 500 ×g for 3 min and used for cytokine profiling. Duplicate wells containing whole blood but treated with 20 µl sterile PBS were used as negative control. Cytokine detection using Proteome Profiler TM arrays Plasma separated from human whole blood stimulated with secretome preparations was applied onto cytokine array membranes for cytokine detection as follows: Nitrocellulose membrane with 36 different capture antibodies spotted in duplicate was used to determine the relative levels of cytokines. Unspecific binding was blocked with assay buffer for 1h at room temperature. The secretome-stimulated PBMC sample (1.5 ml) was diluted in assay buffer with 15 µl of reconstituted human cytokine array detection antibody cocktail and incubated at room temperature for 1h. After washing, the array was treated with streptavidin HRP for 30 min at room temperature on a rocking platform shaker. The array was finally incubated with chemiluminescence reagent for 10 min and images were acquired in Syngene G:Box Imaging System. The positive signals visualized on the array were identified by comparing with the transparency overlay template with the pairs of reference spots in three corners of each array. Mean spot pixel densities were calculated from duplicate spots by analyzing the image using the software provided with G:Box Imaging System. Declarations Acknowledgements This study was supported by Kuwait University Grant SRUL 01/14. We thank the Research Administration of Kuwait University for generous funding to the Oral Microbiology Research Laboratory. Competing interests: The authors declare no competing interests. Author Contributions: MK: Conceived and designed the study, bioinformatics analyses, performed the experiments, manuscript writing RGB: Performed the experiments, manuscript writing MQ: Data interpretation, manuscript review and writing AA: Data interpretation, manuscript review and writing AE: Data interpretation, manuscript review and writing Ethical approval: This study was approved by the ethical committee of the Health Sciences Center, Kuwait University (DR/EC/3413), and has been carried out in full accordance with the World Medical Association Declaration of Helsinki. The blood donor received written information about the nature and purposes of the study and a written informed consent was obtained upon the volunteer’s approval to participate. References 1 Finlay, B. B. & Falkow, S. Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61 , 136-169 (1997). 2 Lee, V. T. & Schneewind, O. Protein secretion and the pathogenesis of bacterial infections. 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Antibiotics (Basel) 9 , doi:10.3390/antibiotics9100654 (2020). 23 Wang, Y., Zhang, W., Wu, Z. & Lu, C. Reduced virulence is an important characteristic of biofilm infection of Streptococcus suis. FEMS Microbiol Lett 316 , 36-43, doi:10.1111/j.1574-6968.2010.02189.x (2011). 24 Terrasse, R., Amoroso, A., Vernet, T. & Di Guilmi, A. M. Streptococcus pneumoniae GAPDH Is Released by Cell Lysis and Interacts with Peptidoglycan. PLoS One 10 , e0125377, doi:10.1371/journal.pone.0125377 (2015). 25 Bem, A. E. et al. Bacterial histidine kinases as novel antibacterial drug targets. ACS Chem Biol 10 , 213-224, doi:10.1021/cb5007135 (2015). 26 Hiratsuka, K., Kiyama-Kishikawa, M. & Abiko, Y. Hemin-binding protein 35 (HBP35) plays an important role in bacteria-mammalian cells interactions in Porphyromonas gingivalis . Microb Pathog 48 , 116-123, doi:10.1016/j.micpath.2010.01.001 (2010). 27 Rainey, K., Michalek, S. M., Wen, Z. T. & Wu, H. Glycosyltransferase-Mediated Biofilm Matrix Dynamics and Virulence of Streptococcus mutans . Appl Environ Microbiol 85 , doi:10.1128/AEM.02247-18 (2019). 28 Ge, X. et al. Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis . PLoS One 11 , e0151142, doi:10.1371/journal.pone.0151142 (2016). 29 Bjur, E., Eriksson-Ygberg, S., Aslund, F. & Rhen, M. Thioredoxin 1 promotes intracellular replication and virulence of Salmonella enterica serovar Typhimurium . Infection and immunity 74 , 5140-5151, doi:10.1128/iai.00449-06 (2006). 30 Kumagai, Y., Yajima, A. & Konishi, K. Peptidase activity of dipeptidyl aminopeptidase IV produced by Porphyromonas gingivalis is important but not sufficient for virulence. Microbiol Immunol 47 , 735-743, doi:10.1111/j.1348-0421.2003.tb03443.x (2003). 31 Kadri, R., Devine, D. & Ashraf, W. Purification and functional analysis of the DnaK homologue from Prevotella intermedia OMZ 326. FEMS Microbiol Lett 167 , 63-68, doi:10.1111/j.1574-6968.1998.tb13208.x (1998). 32 Santos, S. G. et al. Differentially regulated proteins in Prevotella intermedia after oxidative stress analyzed by 2D electrophoresis and mass spectrometry. Anaerobe 18 , 76-82, doi:10.1016/j.anaerobe.2011.12.008 (2012). 33 Guan, S. M. et al. Prevotella intermedia upregulates MMP-1 and MMP-8 expression in human periodontal ligament cells. FEMS Microbiol Lett 299 , 214-222, doi:10.1111/j.1574-6968.2009.01748.x (2009). 34 Kim, S. J. et al. Prevotella intermedia lipopolysaccharide stimulates release of tumor necrosis factor-alpha through mitogen-activated protein kinase signaling pathways in monocyte-derived macrophages. FEMS Immunol Med Microbiol 51 , 407-413, doi:10.1111/j.1574-695X.2007.00318.x (2007). 35 Nath, A., Chattopadhya, S., Chattopadhyay, U. & Sharma, N. K. Macrophage inflammatory protein (MIP)1alpha and MIP1beta differentially regulate release of inflammatory cytokines and generation of tumoricidal monocytes in malignancy. Cancer Immunol Immunother 55 , 1534-1541, doi:10.1007/s00262-006-0149-3 (2006). 36 Caron, E., Gross, A., Liautard, J. P. & Dornand, J. Brucella species release a specific, protease-sensitive, inhibitor of TNF-alpha expression, active on human macrophage-like cells. J Immunol 156 , 2885-2893 (1996). 37 Karched, M., Bhardwaj, R. G., Inbamani, A. & Asikainen, S. Quantitation of biofilm and planktonic life forms of coexisting periodontal species. Anaerobe 35 , 13-20, doi:10.1016/j.anaerobe.2015.04.013 (2015). 38 Deatherage Kaiser, B. L. et al. Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins. J Microbiol Methods 118 , 18-24, doi:10.1016/j.mimet.2015.08.008 (2015). 39 Karched, M., Bhardwaj, R. G., Tiss, A. & Asikainen, S. Proteomic Analysis and Virulence Assessment of Granulicatella adiacens Secretome. Front Cell Infect Microbiol 9 , 104, doi:10.3389/fcimb.2019.00104 (2019). 40 Alkandari, S. A., Bhardwaj, R. G., Ellepola, A. & Karched, M. Proteomics of extracellular vesicles produced by Granulicatella adiacens , which causes infective endocarditis. PLoS One 15 , e0227657, doi:10.1371/journal.pone.0227657 (2020). 41 Hiller, K., Schobert, M., Hundertmark, C., Jahn, D. & Munch, R. JVirGel: Calculation of virtual two-dimensional protein gels. Nucleic Acids Res. 31 , 3862-3865 (2003). 42 Yu, N. Y. et al. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26 , 1608-1615, doi:10.1093/bioinformatics/btq249 (2010). 43 Almagro Armenteros, J. J. et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 37 , 420-423, doi:10.1038/s41587-019-0036-z (2019). 44 Bagos, P. G., Tsirigos, K. D., Liakopoulos, T. D. & Hamodrakas, S. J. Prediction of lipoprotein signal peptides in Gram-positive bacteria with a Hidden Markov Model. J. Proteome Res. 7 , 5082-5093, doi:10.1021/pr800162c (2008). 45 Bendtsen, J. D., Nielsen, H., Widdick, D., Palmer, T. & Brunak, S. Prediction of twin-arginine signal peptides. BMC Bioinformatics 6 , 167, doi:10.1186/1471-2105-6-167 (2005). 46 Conesa, A. et al. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21 , 3674-3676, doi:10.1093/bioinformatics/bti610 (2005). 47 Yu, C. S. et al. CELLO2GO: a web server for protein subCELlular LOcalization prediction with functional gene ontology annotation. PLoS One 9 , e99368, doi:10.1371/journal.pone.0099368 (2014). 48 von Mering, C. et al. STRING: known and predicted protein-protein associations, integrated and transferred across organisms. Nucleic Acids Res. 33 , D433-437, doi:10.1093/nar/gki005 (2005). 49 Garg, A. & Gupta, D. VirulentPred: a SVM based prediction method for virulent proteins in bacterial pathogens. BMC Bioinformatics 9 , 62, doi:10.1186/1471-2105-9-62 (2008). Additional Declarations No competing interests reported. Supplementary Files SupplFileS1PiBiofilmProteinslist.xlsx SupplFileS2PiPlanktonicProteinslist.xlsx Cite Share Download PDF Status: Published Journal Publication published 04 Apr, 2022 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 27 Jan, 2022 Reviews received at journal 25 Jan, 2022 Reviews received at journal 09 Dec, 2021 Reviewers agreed at journal 03 Dec, 2021 Reviewers invited by journal 03 Dec, 2021 Editor assigned by journal 03 Dec, 2021 Editor invited by journal 03 Dec, 2021 Submission checks completed at journal 03 Dec, 2021 First submitted to journal 14 Nov, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1078670","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":67643483,"identity":"5047f658-8960-4029-a759-01ed0b9856ef","order_by":0,"name":"Maribasappa Karched","email":"data:image/png;base64,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","orcid":"","institution":"Kuwait University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Maribasappa","middleName":"","lastName":"Karched","suffix":""},{"id":67643484,"identity":"99f62f9c-cdf6-416b-94d4-ea2f36afb86b","order_by":1,"name":"Radhika Bhardwaj","email":"","orcid":"","institution":"Kuwait University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Radhika","middleName":"","lastName":"Bhardwaj","suffix":""},{"id":67643485,"identity":"86bd28e5-08b7-44c6-a510-3fafb27851e4","order_by":2,"name":"Muawia Qudeimat","email":"","orcid":"","institution":"Kuwait University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muawia","middleName":"","lastName":"Qudeimat","suffix":""},{"id":67643486,"identity":"1c36de55-cf4d-4be4-ba20-a616e46ad5ba","order_by":3,"name":"Areej Al-Khabbaz","email":"","orcid":"","institution":"Kuwait University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Areej","middleName":"","lastName":"Al-Khabbaz","suffix":""},{"id":67643487,"identity":"9629eae6-3980-46dc-be8f-047fb9ab5fbc","order_by":4,"name":"Arjuna Ellepola","email":"","orcid":"","institution":"Kuwait University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Arjuna","middleName":"","lastName":"Ellepola","suffix":""}],"badges":[],"createdAt":"2021-11-14 09:44:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1078670/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1078670/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-022-09085-0","type":"published","date":"2022-04-04T18:44:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":16219672,"identity":"29c250a1-6152-439d-8fd2-eaba13ae92b4","added_by":"auto","created_at":"2021-12-06 17:13:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77539,"visible":true,"origin":"","legend":"Analysis of the proteome of P. intermedia biofilm and planktonic cells. (A) SDS-PAGE gel showing protein bands from protein preparations: biofilm (lane 1, 2) and planktonic cells (lane 3, 4). (B) Western blot analysis of the secretome preparations (lane 1=biofilm, lane 2= planktonic) and the WCP (lane 3) using an antibody for the cytoplasmic marker protein FtsZ. (C) Protein sequences from LC-MS analysis of the secretome were analyzed by an in silico 2DE tool.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1078670/v1/fe8485dd31ec2ee3ba153934.jpg"},{"id":16219538,"identity":"910a0491-0fda-4b1c-a6cf-f265baa764b0","added_by":"auto","created_at":"2021-12-06 17:10:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41346,"visible":true,"origin":"","legend":"Subcellular localization of proteins. Protein FASTA sequences were analyzed for their subcellular localization using the bioinformatics tool CELLO2GO. The results obtained were compared with other predictions tools such as PSORTb and SignalP.","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1078670/v1/3640cf6f81e1134f4a7e06ae.jpg"},{"id":16219673,"identity":"86922f52-da37-4d30-9f17-036283312069","added_by":"auto","created_at":"2021-12-06 17:13:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":94916,"visible":true,"origin":"","legend":"Gene Ontology analysis of P. intermedia proteomes from biofilm and planktonic cells. Gene ontology annotation was achieved using Blast2GO and an online software “CELLO2GO”. Protein sequences were grouped into 3 categories based on their properties and functions. ","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1078670/v1/ef4178fbc334af6899845cb6.jpg"},{"id":16219544,"identity":"8e1a6b8d-46c7-4d7e-b1cb-fe4d84f4d7a3","added_by":"auto","created_at":"2021-12-06 17:10:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":181409,"visible":true,"origin":"","legend":"Functional protein association networks of P. intermedia proteome from biofilm and planktonic cells. The online tool STRING was used for grouping the secreted proteins based on functional networks. Minimum interaction scores were set at a strong confidence level of 0.7. The three major network groups formed are shown in dotted circles. Seven different colored link several nodes and represent seven types of evidence used in predicting associations. A red line indicates the presence of fusion evidence; a green line represents neighborhood evidence; a blue line represents co-occurrence evidence; a purple line represents experimental evidence; a yellow line represents text-mining evidence; a light blue line represents database evidence and a black line represents co-expression evidence.","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1078670/v1/62737aa2d48b070e2e5a63e7.jpg"},{"id":16219543,"identity":"895a732d-8770-427b-8373-001afd946f72","added_by":"auto","created_at":"2021-12-06 17:10:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59270,"visible":true,"origin":"","legend":"Cytokine induction from human whole blood by P. intermedia biofilm and planktonic cells. Human whole blood collected from a healthy volunteer was stimulated by secretome preparations from the biofilms and planktonic cultures of P. intermedia for 24 h. The cytokines produced were detected by using Proteome ProfilerTM membrane array. Means (SD) of signal densities of spots were determined using Gene Tools analysis software in Syngene Imaging System.","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1078670/v1/6d248a4c8ea5357eef1de08b.jpg"},{"id":19946849,"identity":"86dc7276-8e09-4762-bdb5-a9afff6c1304","added_by":"auto","created_at":"2022-04-04 18:44:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":932731,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1078670/v1/64a60194-8c6f-4f5f-9cd4-b44fbb0b3525.pdf"},{"id":16219539,"identity":"08b171a7-4cc5-477d-80a3-a1afc20539b7","added_by":"auto","created_at":"2021-12-06 17:10:09","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25062,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFileS1PiBiofilmProteinslist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1078670/v1/3c070b075666c7c2e684bd7a.xlsx"},{"id":16219542,"identity":"aca08122-cd3d-4bbc-bbd5-7b62af325ee9","added_by":"auto","created_at":"2021-12-06 17:10:09","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23470,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFileS2PiPlanktonicProteinslist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1078670/v1/caf05e63ce490d358a0bbeb1.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eProteomic Analysis of the Periodontal Pathogen Prevotella Intermedia Secretomes in Biofilm and Planktonic Lifestyles\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBacteria release proteins and other components into extracellular milieu continuously during normal growth and physiology. Many of the secreted proteins often function in nutrient acquisition, but in pathogenic bacteria, they play a key role in disease, e.g., by helping the bacteria in host colonization or by modulating host immune responses \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Thus, extracellular secretion of bacterial proteins is an important virulence mechanism. To achieve this, bacteria have devised various strategies, e.g., dedicated secretory systems \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and extracellular vesicles \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The central components of the main protein translocation system, the Sec system, share a high degree of sequence similarity between Gram-positive and Gram-negative bacteria.\u003c/p\u003e \u003cp\u003eOral infectious diseases such as caries and periodontitis are dental plaque biofilm-driven\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Dental plaque is a structurally and functionally organized, highly complex multispecies biofilm. The resident bacteria in this multispecies community exhibit extensive interactions while forming bioଁlm structures, carrying out physiological functions, and inducing microbial pathogenesis \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Interspecies interactions in biofilms are competitive, cooperative and, antagonistic \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To facilitate such interactions, plaque bacteria may release several cellular components into the extracellular space within the biofilm matrix. In addition, proteins secreted in biofilms may have specific effects in terms of the virulence properties of bioଁlm residents, which could inଂuence the overall pathogenicity of biofilms.\u003c/p\u003e \u003cp\u003ePeriodontitis is a chronic inflammatory disease characterized by mild to moderately severe inflammation of the periodontal tissue, progressive destruction of ligament fibers, as well as alveolar bone loss \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The disease is primarily related to chronic plaque accumulation in a susceptible host. Major bacterial species implicated in periodontitis are, \u003cem\u003ePorphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans\u003c/em\u003e and others \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePrevotella intermedia\u003c/em\u003e occurs frequently in the subgingival samples of periodontitis patients \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Further, presence of \u003cem\u003ePrevotella\u003c/em\u003e has been positively correlated with clinical attachment loss \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, bleeding on probing \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and periodontal \u003ca class=\"FNLink\" href=\"#Fn2\" id=\"#FNLinkFn2\"\u003e\u003c/a\u003einflammation \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eP. intermedia\u003c/em\u003e is a Gram-negative, non-motile, rod-shaped, bacterium that requires strict anaerobic conditions for growth. In addition to oral infections, \u003cem\u003eP. intermedia\u003c/em\u003e has also been detected from nonoral sites, e.g., NOMA (cancrum oris) lesions \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and bacterial tracheitis in children \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Further, chronic oral infections such as periodontitis in which \u003cem\u003eP. intermedia\u003c/em\u003e is a major species, increase the risk of systemic diseases, such as atherosclerosis, pre-term delivery of low birth-weight infants \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Importantly, \u003cem\u003eP. intermedia\u003c/em\u003e is known to be resistant to several antibiotics including cephalosporins, penicillins and tetracyclins \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Since \u003cem\u003eP. intermedia\u003c/em\u003e is not an exogenous pathogen and is a part of normal oral microbiota, its complete elimination is not possible. Despite the remarkable clinical significance \u003cem\u003eP. intermedia\u003c/em\u003e has, little is known about the molecular basis for its virulence. For \u003cem\u003eP. intermedia\u003c/em\u003e to survive in a complex and competitive oral environment, it is imperative that it can adhere to surfaces and integrate into plaque biofilm. In general, bacterial cells dispersed from mature plaque biofilms are collected by saliva and can be regarded as planktonic cells. Such planktonic bacterial cells can reattach to oral surfaces and initiate new biofilm growth. Recent knowledge from the literature suggests that virulence potentials of the biofilms and the planktonic cells are remarkably different \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Previously, even though different aspects of \u003cem\u003eP. intermedia\u003c/em\u003e as part of plaque biofilm have been studied, extracellular proteins secreted by this species (secretome), in biofilm or in planktonic life form, has not been investigated.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAnalysis of the secretomes of \u003cem\u003eP. intermedia\u003c/em\u003e biofilm and planktonic cells\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein preparations (Fig. 1) from \u003cem\u003eP. intermedia\u003c/em\u003e were analyzed by LC-MS/MS. Database search (NCBI-nr) revealed 109 proteins from the biofilm (Suppl. File S1) and 136 proteins from planktonic cells (Suppl. File S2). To ensure that the secretome preparations from the biofilms and the planktonic cells did not contain cytoplasmic proteins due to cell lysis, western blot analysis was performed using an antibody against a cytoplasmic marker protein FtsZ. Panel B in Fig 1 shows the presence of the marker protein from the whole cell protein preparation from \u003cem\u003eP. intermedia\u003c/em\u003e, but not from the secretome preparations. As depicted in a theoretical 2DE map of the secretome, the MW of the secreted proteins ranged between 5 kDa and 130 kDa (Fig. 1C). In both biofilm and planktonic cells, with respect to predicted pI values, majority of the proteins formed a cluster with the pI range of 4.0 and 6.5.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtein sequences of the secretomes of \u003cem\u003eP. intermedia\u003c/em\u003e biofilms and planktonic cells were analyzed for the route of their extracellular release by various bioinformatics tools. Biofilm and planktonic preparations showed 17 and 33 signal peptide-bearing, 13 and 18 lipobox bearing and 2 and 0 TatP signal bearing proteins respectively. Transmembrane alpha helices were found in 4 and 6 proteins from biofilm and planktonic preparations. Subcellular localization analysis (Fig. 2) revealed that the secretome from planktonic cells contained more extracellular proteins (11.6%) than the biofilms (7.4%). Proteins of cytoplasmic origin were more in the biofilm (56.4%) than the planktonic cells (50%). Further, 40-50% of the proteins identified from the secretomes were unique to biofilm or planktonic cells. Importantly, proteins such as superoxide reductase, sensor histidine kinase, C40 family peptidase, elongation factor Tu, threonine synthase etc. were unique to biofilm secretome and were not detected in the planktonic secretome.\u003c/p\u003e\n\u003cp id=\"isPasted\"\u003e\u003cstrong\u003ePotential virulence proteins in \u003cem\u003eP. intermedia\u003c/em\u003e secretome.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVirulence potential of the \u003cem\u003eP. intermedia\u003c/em\u003e was assessed by \u003cem\u003ein silico\u003c/em\u003e prediction of virulence factors using the online tools \u0026ldquo;VirulentPred\u0026rdquo; and \u0026ldquo;VFDB\u0026rdquo; (Virulence Factor DataBase). We found that 31 proteins from the biofilm secretome and 30 proteins from planktonic secretome were predicted to be virulent (Tables 1 and 2). Hemin-binding protein, porin family protein, OmpA, thioredoxin, Omp-28, tetratricopeptide-binding protein molecular chaperones DnaK and GroES, were the major proteins with demonstrated virulence properties in other bacterial species.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Proteins with predicted virulence properties from \u003cem\u003eP. intermedia\u003c/em\u003e biofilm.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference Number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_014710387.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eDNA starvation/stationary phase protection protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028905462.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028905462.1\"\u003eWP_028905462.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003ePeptidase M6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028905527.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028905527.1\"\u003eWP_028905527.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eSensor histidine kinase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028905084.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028905084.1\"\u003eWP_028905084.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_WP_028905084\" title=\"Go to alignment for tetratricopeptide repeat protein [Prevotella intermedia] \u003egb|APW32430.1| hypothetical protein BWX39_07160 [Prevotella intermedia ATCC 25611 = DSM 20706]\"\u003eTetratricopeptide repeat protein\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028905748.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028905748.1\"\u003eWP_028905748.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003ehemin-binding protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_014710403.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_014710403.1\"\u003eWP_014710403.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003ethiol reductase thioredoxin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_004356500.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eXaa-Pro aminopeptidase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_004364886.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003etrypsin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028905224.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028905224.1\"\u003eWP_028905224.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003epeptidyl-prolyl cis-trans isomerase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_013265509.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eNADH oxidase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028904949.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028904949.1\"\u003eWP_028904949.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003epeptide ABC transporter substrate-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_018667829.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eATPase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_015531502.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eMFS transporter\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_172460529.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_172460529.1\"\u003eWP_172460529.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_WP_172460529\" title=\"Go to alignment for ROK family protein [Prevotella intermedia]\"\u003eROK family protein\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028904772.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028904772.1\"\u003eWP_028904772.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eurocanate hydratase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028904668.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028904668.1\"\u003eWP_028904668.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eribulose-phosphate 3-epimerase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_007835729.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eAsp/Glu/hydantoin racemase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_005332057.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eN-acetylmuramoyl-L-alanine amidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_007133390.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eMolecular \u0026nbsp;chaperone DnaJ\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003eMBP5257375.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eacetyltransferase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028906355.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028906355.1\"\u003eWP_028906355.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_WP_028906355\" title=\"Go to alignment for substrate-binding domain-containing protein [Prevotella intermedia] \u003egb|APW31683.1| AraC family transcriptional regulator [Prevotella intermedia ATCC 25611 = DSM 20706]\"\u003esubstrate-binding domain-containing protein\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028904901.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028904901.1\"\u003eWP_028904901.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eenergy transducer TonB\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003eWP_007411110.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_MBR2101597\" title=\"Go to alignment for anthranilate phosphoribosyltransferase [Prevotella sp.]\"\u003eanthranilate phosphoribosyltransferase\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028906371.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028906371.1\"\u003eWP_028906371.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003e1-acyl-sn-glycerol-3-phosphate acyltransferase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/MBO5313912.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=EDBANTRR013\" title=\"Show report for MBO5313912.1\"\u003eMBO5313912.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eMembrane protein M15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028905881.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028905881.1\"\u003eWP_028905881.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003emultidrug ABC transporter ATP-binding protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/MBF1618150.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=EDBANTRR013\" title=\"Show report for MBF1618150.1\"\u003eMBF1618150.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_MBF1618150\" title=\"Go to alignment for type IV secretion protein Rhs [Prevotella sp.]\"\u003etype IV secretion protein Rhs\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_028906306.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_028906306.1\"\u003eWP_028906306.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eL-asparaginase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/WP_014709317.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=E80BV82D013\" title=\"Show report for WP_014709317.1\"\u003eWP_014709317.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003eYkgB family protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/MBQ7451373.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=EDBANTRR013\" title=\"Show report for MBQ7451373.1\"\u003eMBQ7451373.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003ethreonine synthase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"22.6984126984127%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/protein/MBP3838531.1?report=genbank\u0026log$=prottop\u0026blast_rank=1\u0026RID=EDBANTRR013\" title=\"Show report for MBP3838531.1\"\u003eMBP3838531.1\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"77.3015873015873%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_MBP3838531\" title=\"Go to alignment for HAD-IA family hydrolase [Prevotella sp.]\"\u003eHAD-IA family hydrolase\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Proteins with predicted virulence properties from \u003cem\u003eP. intermedia\u003c/em\u003e planktonic cells.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable align=\"left\" border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference Number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_014710403.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003ethiol reductase thioredoxin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_028905059.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003etrypsin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_025000944.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eDNA starvation/stationary phase protection protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_014709654.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003epeptidase M6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_028906361.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eDNA topoisomerase II\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_028905748.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003ehemin-binding protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_028905169.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eDUF4595 domain-containing protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_014709619.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eenoyl-ACP reductase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_028905189.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003ePorT family protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBR7087708.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eamino acid adenylation domain-containing protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBP7359878.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003echemotaxis protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBO7539992.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_MBO7539992\" title=\"Go to alignment for DNA polymerase III subunit gamma/tau [Prevotella sp.]\"\u003eDNA polymerase III subunit gamma/tau\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBQ3767790.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eATP-binding protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_014709212.1|\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003epeptidylprolyl isomerase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBO7578384.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eTolC family protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_014709366.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eABC transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBP8758149.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_MBP8758149\" title=\"Go to alignment for DUF1622 domain-containing protein [Prevotella sp.]\"\u003eDUF1622 domain-containing protein\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBR2882634.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_MBR2882634\" title=\"Go to alignment for ankyrin repeat domain-containing protein [Prevotella sp.]\"\u003eankyrin repeat domain-containing protein\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_099836288.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eDNA-binding response regulator\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_097549978.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_WP_097549978\" title=\"Go to alignment for tRNA epoxyqueuosine(34) reductase QueG [Prevotella intermedia] \u003egb|PDP60535.1| tRNA epoxyqueuosine(34) reductase QueG [Prevotella intermedia]\"\u003etRNA epoxyqueuosine(34) reductase QueG\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBA7488061.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_MBA7488061\" title=\"Go to alignment for calcineurin-like phosphoesterase C-terminal domain-containing protein [Prevotella sp.]\"\u003ecalcineurin-like phosphoesterase C-terminal domain-containing protein\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBP5424796.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003esigma-70 family RNA polymerase sigma factor\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_100190220.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003epeptidase M20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_100356678.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_WP_100356678\" title=\"Go to alignment for type IV secretion protein Rhs [Prevotella intermedia] \u003egb|PJI24114.1| type IV secretion protein Rhs [Prevotella intermedia]\"\u003etype IV secretion protein Rhs\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_088437864.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eABC transporter ATP-binding protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_099984831.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eglycosyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eMBP9983829.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eMarR family transcriptional regulator\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_099976545.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003e\u003ca href=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_WP_099976545\" title=\"Go to alignment for HAMP domain-containing histidine kinase [Prevotella intermedia] \u003egb|PIN28473.1| two-component sensor histidine kinase [Prevotella intermedia]\"\u003eHAMP domain-containing histidine kinase\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eRKW57308.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eSDR family oxidoreductase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"21.21212121212121%\"\u003e\n \u003cp\u003eWP_097656281.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"78.78787878787878%\"\u003e\n \u003cp\u003eTonB-dependent receptor\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eGene ontology analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO) analysis of the amino acid FASTA sequences of the \u003cem\u003eP. intermedia\u003c/em\u003e secretomes was achieved by using the tools Blast2GO and CELLO2GO. As shown in Fig. 3,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emarked differences in the percentage of proteins was found with GO annotations in \u0026ldquo;biological processes\u0026rdquo; and \u0026ldquo;molecular functions\u0026rdquo;. In the category biological processes, biofilm secretome showed higher number of proteins in the case of catabolic process, pathogenesis, cellular amino acid metabolic process, small molecule metabolic process, cellular nitrogen compound metabolic process, while proteins in the \u0026ldquo;transport\u0026rdquo; group were higher in planktonic secretome. In the category \u0026ldquo;molecular function\u0026rdquo;, biofilm showed higher proteins with \u0026ldquo;protein binding\u0026rdquo;, \u0026ldquo;methyltransferase\u0026rdquo; and \u0026ldquo;kinase\u0026rdquo; activities. The planktonic secretome showed higher number of proteins with peptidase activity.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"isPasted\"\u003e\u003cstrong\u003eFunctional protein association network analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs seen in Fig. 4, \u003cem\u003eP. intermedia\u003c/em\u003e secretome proteins formed three major groups in the STRING network, i.e., carbohydrate metabolism, ribosomal proteins, and chaperones/virulence proteins. Components of the sugar metabolism network were glutamate dehydrogenase, glucose-6-phosphate isomerase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphate aldolase, serine hydroxymethyltransferase, and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePutative virulence-associated proteins and molecular chaperones such as thioredoxin, DnaK, dps, and GroEL formed another cluster. The ribosomal protein cluster included rplF, rpsE, rpsM, rpsB rSA2, and rplL5 in both biofilm and planktonic cells (Fig. 4).\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"isPasted\"\u003e\u003cstrong\u003eInflammatory potential of the \u003cem\u003eP. intermedia\u003c/em\u003e secretomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo get a preliminary insight into the inflammatory potential, human whole blood was stimulated with \u003cem\u003eP. intermedia\u003c/em\u003e secretomes from biofilm and planktonic cultures. As determined by signal densities of cytokine spots on a membrane array (Fig. 5), secretome preparations from both biofilm and planktonic cells induced similar levels of CCL5/RANTES, ICAM-1, MIF, and Serpin E1. The biofilm secretome additionally induced cytokines MIP-1a/MIP-1b, IL-1b, and IL-8.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eProtein secretion is a fundamental physiological process among bacteria. However, the components of the secretome may vary depending on the niche where the bacteria are located. Biofilm life is a modus operandi for most oral bacteria. Bacterial residents of plaque biofilms continuously release cellular components into the extracellular environment in the biofilm. Interestingly, in this study, proteins such as superoxide reductase, sensor histidine kinase, C40 family peptidase, elongation factor Tu, threonine synthase, which are known to play important roles in bacterial virulence and colonization in the host, were unique to biofilm secretome. Thus, fundamental differences, including those with respect to protein secretion, may exist between the biofilm and planktonic mode of life.\u003c/p\u003e\n\u003cp\u003eThe possibility of contamination of the secretome preparations with subcellular proteins resulting from cell lysis was ruled out by using an established cytoplasmic marker protein Fts-Z \u003csup\u003e24\u003c/sup\u003e which was undetected in all \u003cem\u003eP. intermedia\u003c/em\u003e secretome preparations.\u003c/p\u003e\n\u003cp\u003ePotential virulence factors in the secretome preparations were predicted by \u003cem\u003ein silico\u003c/em\u003e analysis. While the total number of predicted virulence factors did not differ between the two secretomes (biofilm=31, planktonic=30), only 6 proteins, including thioredoxin, trypsin and hemin-binding protein, were common between them. This is an interesting finding given the fundamental differences of bacteria in biofilm and planktonic modes of life. In the subgingival environment where \u003cem\u003eP. intermedia\u003c/em\u003e is part of the plaque biofilm encountering a multitude of host challenges, it probably needs to secrete an entirely different set of proteins with virulence potential compared to the planktonic form of life. Of noteworthy virulence factors in the biofilm secretome were thioredoxin, hemin-binding protein, peptidase M6, NADH oxidase and trypsin. In the case of planktonic secretome, enoyl-ACP reductase, PorT family protein, peptidase M20, and glycosyltransferase were some of the important ones. Interestingly, histidine kinases, which were found in both biofilm and planktonic secretomes, are recently proposed to be attractive antibacterial drug targets \u003csup\u003e25\u003c/sup\u003e. Further, hemin-binding protein that plays a role in bacterial interaction with host cells \u003csup\u003e26\u003c/sup\u003e\u003cstrong\u003e, glycosyltransferases that mediate biofilm\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eformation\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e27\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSimilarly, virulence potential of other proteins such as NADH oxidase \u003csup\u003e28\u003c/sup\u003e and thioredoxin \u003csup\u003e29\u003c/sup\u003e\u0026nbsp; has been well established in several other bacterial pathogens.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo get an insight into the functional significance of the proteins identified from the \u003cem\u003eP. intermedia\u003c/em\u003e proteomes, gene ontology analysis was carried out on the fasta sequences. The biofilm secretome contained higher percentage of proteins in \u0026ldquo;biological processes\u0026rdquo; category with catabolic process, cellular amino acid metabolic process, cellular nitrogen compound process. Biofilm secretome also showed higher number of proteins with protein binding, methyl transferase and kinase activities. Interestingly, the planktonic secretome appeared to possess higher percentage of proteins with peptidase activity, compared to the biofilm secretome. In another important periodontal pathogen \u003cem\u003eP. gingivalis\u003c/em\u003e, certain peptidases were important for the survival of the species but not sufficient for its virulence\u0026nbsp;\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe biofilm and planktonic secretomes were analyzed for their functional network associations by using STRING. Three function-based groups were identified, carbohydrate metabolism, ribosomal proteins and a third network comprising of virulence factors and chaperones. Key enzymes in carbohydrate metabolism, glutamate dehydrogenase, glucose-6-phosphate isomerase and phosphoenolpyruvate carboxylase were part of the network. Predicted virulence factors that also function as molecular chaperones, such as thioredoxin, DnaK, dps and GroEL formed a distinct network. Several years ago, the ability of \u003cem\u003eP. intermedia\u003c/em\u003e DnaK to function as a molecular chaperone was assessed by a luciferase folding assay\u0026nbsp;\u003csup\u003e31\u003c/sup\u003e. In periodontitis, stresses such as temperature and pH increase, increased flow of gingival crevicular fluid, and oxidative stress resulting from phagocytic cells. In response to these stresses, \u003cem\u003eP. intermedia\u003c/em\u003e may show increased expression of heat-shock proteins such as DnaK. This may contribute to enhanced virulence of the species as well as its survival in stress conditions. In a study that investigated the effect of oxidative stress on \u003cem\u003eP. intermedia\u003c/em\u003e protein exopression\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e\u003cstrong\u003e,\u003c/strong\u003e several proteins from the above functional networks, including fructose-1, 6-bisphosphate aldolase, reductases, ribosomal proteins and molecular chaperones like thioredoxin, DnaK were upregulated when bacteria were exposed to molecular oxygen. The significance of the secretion of these proteins may possibly be related to the ability of \u003cem\u003eP. intermedia\u003c/em\u003e to travel beyond the oral cavity and colonize distant body sites where the species must adapt to oxygenated tissues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. intermedia\u003c/em\u003e has been considered a major periodontal pathogen because of its established role in initiation and development of periodontitis by inducing a variety of proinflammatory cytokines, proteases, and matrix metalloproteinases. Important cytokines such as IL-8, IL1-b, and macrophage inflammatory proteins were induced by the biofilm secretome. \u003cem\u003eP. intermedia\u003c/em\u003e and its components have been shown to induce IL-8, IL1-b, TNF-a\u0026nbsp;and MMPs\u0026nbsp;\u003csup\u003e33,34\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;The role of these and other proinflammatory cytokines such as MIP-\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and MIP-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;in periodontitis has been well established\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e35\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e. Importantly, certain bacteria-secreted proteins stimulate cytokines only in their secreted-form, not when they are within the bacterial cell\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e36\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e. This type of specificity may suggest possible roles of \u003cem\u003eP. intermedia\u003c/em\u003e secretome proteins beyond the oral cavity.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn conclusion, the current proteomics data on the biofilm- and planktonic- secretomes may provide new insights into virulence mechanisms of \u003cem\u003eP. intermedia\u003c/em\u003e. For example, the role/potential of the predicted virulence factors in the secretomes may be studied by overexpressing the respective genes in a suitable system, and by constructing specific knockout mutants for further studies using \u003cem\u003ein vitro\u003c/em\u003e and/or \u003cem\u003ein vivo\u003c/em\u003e models.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eBiofilm and planktonic cultures\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. intermedia\u003c/em\u003e ATCC 25611 was grown on brucella blood agar containing 5% sheep blood in anaerobic atmosphere at 37\u0026nbsp;\u0026deg;C for 3 days. Biofilms and planktonic cultures were grown as described earlier with some modifications.\u003csup\u003e37\u003c/sup\u003e Bacterial colonies were harvested from agar plates with sterile disposable loops and suspended in brucella broth. The bacterial cells were washed at least once by suspending in brucella broth and then collected by centrifugation at 5000\u0026times;g for 5 min. The washed bacterial cell pellet was resuspended in 1 ml brucella broth to make a stock suspension. \u0026nbsp;A final bacterial suspension of OD\u003csub\u003e600\u003c/sub\u003e=1 was prepared after measuring the optical density of the stock suspension.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBiofilms and broth cultures for planktonic bacterial growth were initiated by inoculating 24-well plates and microfuge tubes, respectively, containing 900 \u0026micro;l brucella broth with a 100-\u0026micro;l aliquot from an OD\u003csub\u003e600\u003c/sub\u003e=1 suspension of each species. Wells or tubes with only broth were considered as negative control. \u0026nbsp;The plates/tubes were incubated in the same culture conditions as above for 24 h. At the end of incubation period, supernatant broth from biofilms was aspirated and the supernatants from planktonic cultures were collected by centrifugation. These supernatants were subjected to secretome preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of secretome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe secretomes were prepared by extracting proteins using tri-chloroacetic acid (TCA) precipitation method as described previously with modifications \u003csup\u003e38,39\u003c/sup\u003e. TCA stock (100% w/v) was mixed with supernatant culture broth at 1:4 ratio and incubated for 30 min at -20 \u003csup\u003e\u0026deg;\u003c/sup\u003eC. After centrifugation at 14,000 \u0026times;g for 20 min at 4 \u003csup\u003e\u0026deg;\u003c/sup\u003eC, traces of acid in the pellet were removed by washing twice with 0.5 ml cold acetone, followed by complete air-drying in a fume hood. The samples were desalted by ultrafiltration through 3K Ultra-0.5 centrifugal filter devices (Amicon) at 14,000 \u0026acute;g for 15 min at 4 \u003csup\u003e\u0026deg;\u003c/sup\u003eC. After discarding the flow-through, concentrates in the columns were finally eluted from columns by centrifugation at 1000 \u0026acute;g for 2 min at 4 \u003csup\u003e\u0026deg;\u003c/sup\u003eC. Broth without bacteria was incubated in parallel and used as negative control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole cell protein preparation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. intermedia\u003c/em\u003e colonies harvested from BBA plates were washed in sterile PBS by centrifugation at 5000 \u0026times;g for 5 min. The pellet was resuspended in lysis buffer containing 1 mg/ml lysozyme and 1 mM phenyl methyl sulfonyl fluoride (PMSF) and incubated for 4 h at 4 - 8\u0026nbsp;\u003csup\u003e\u0026deg;\u003c/sup\u003eC. The samples were subject to sonication in Omni Ruptor at a pulse rate 40 for 8 times (1 min sonication with 1 min interval on ice). The lysates were centrifuged at 10,000 \u0026times;g for 10 min at 4\u0026nbsp;\u003csup\u003e\u0026deg;\u003c/sup\u003eC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Protein Concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein concentrations in secretome preparations and whole cell lysates were estimated by Quick Start\u003csup\u003eTM\u003c/sup\u003e Bradford protein microplate standard assay (Bio-Rad) as per manufacturer instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS-PAGE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein samples were mixed with 5\u0026times; Laemmli sample buffer (125 mM tris, pH 6.8; 6 % glycerol, 2 % SDS; 5 % beta-mercapthoethanol; 0.025 % bromophenol blue) and boiled at 95 \u0026deg;C for 5 minutes. The samples were loaded on a 15 % SDS-PAGE gel [4 % stacking gel (4 % acrylamide; 68 mM tris, pH 6.8; 0.2 % SDS), 15 % separating gel; 375 mM tris, pH 8.8; 0.1 % SDS]. Electrophoresis was run at 150 V for 75 minutes (Mini-protein II Dual Slab Cell, Bio Rad) and the protein bands were visualized using coommassie blue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ensure that the secretome preparations did not contain proteins that originated due to cell lysis, western blot analysis of whole cell lysate and secretome preparations was performed. Protein bands on the gel were transferred onto a PVDF membrane using Trans-Blot\u003csup\u003e\u0026reg;\u003c/sup\u003e Turbo\u003csup\u003eTM\u003c/sup\u003e transfer system (Bio-Rad). To avoid unspecific binding, membrane was blocked with 5% skimmed milk overnight at 4\u003csup\u003e\u0026deg;\u003c/sup\u003eC. As primary antibody, an antibody against the cytoplasmic marker protein, Ftsz (Agrisera AB, Sweden) was used at 1:1000 dilution and incubated 1 h at room temperature. The membrane was then incubated as above with a peroxidase conjugated goat antirabbit secondary antibody (1: 5000). The membrane was washed with tris-buffer saline containing Tween-20 (TBST). Finally, the bound antibodies on the membrane were detected by using SuperSignal\u003csup\u003eTM\u003c/sup\u003e West Pico chemiluminescence substrate (Pierce) and images were acquired in G:Box Imaging System (Syngene).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of proteins by Nano-LC-ESI-MS/MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the identification of proteins in the secretomes from biofilms and planktonic cultures, mass spectrometry was performed at Proteome Factory (Proteome Factory AG, Berlin, Germany) using nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With an Agilent 1100 nanoHPLC system (Agilent, Waldbronn, Germany) interfaced to an Orbitrap Velos (Thermo Scientific, Bremen, Germany) via a nanoelectrospray ion source. After pooling replicate samples from EVs preparations, proteins were reduced, alkylated and digested by trypsin (Promega, Mannheim, Germany). Then, 400 ng of the resulting peptides were subjected to the nanoLC-ESI-MS/MS. 1% acetonitrile/0.5% formic acid was used as eluent for 5 minutes to trap and desalt the peptides on the enrichment column (Zorbax 300SB-C18, 0.3\u0026nbsp;\u0026acute;\u0026nbsp;5 mm, Agilent). A water/acetonitrile (both supplemented with 0.1% formic acid) gradient from 5% to 40% acetonitrile was then used within 120 minutes to separate the peptides on a Zorbax 300SB-C18, 75 \u0026micro;m x 150 mm column (Agilent). The mass spectrometer automatically recorded mass spectra, and tandem mass spectra were data-dependently acquired for multiply charged ions. Protein identification was made using the Mascot search engine (Matrix Science, London, England) against the bacterial subset of the RefSeq protein database (National Center for Biotechnology Information), (downloaded on 1\u003csup\u003est\u003c/sup\u003e July 2016, 49867978 entries,\u0026nbsp;NCBI, Bethesda, USA) and a database with common protein contaminants. For MS/MS spectra where assignment of the precursor ion\u0026rsquo;s charge state was missing,\u0026nbsp;search parameters for ions from ESI-MS/MS data acquisition was set to \u0026quot;2+, 3+ or 4+\u0026quot;\u0026nbsp;according to the instrument\u0026apos;s and\u0026nbsp;method\u0026apos;s standard charge state distribution. The search parameters were: Fixed modifications: Carbamidomethyl (C); variable modifications: Deamidated (NQ), Oxidation (M); Peptide Mass Tolerance: \u0026plusmn; 3 ppm; Fragment Mass Tolerance: \u0026plusmn; 0.6 Da;\u0026nbsp;Missed Cleavages: 2.\u0026nbsp;The inclusion criterion was: peptides that match with a score of 20 or above. Mass spectrometry data, with the project acession number PXD029419, has been deposited at PRIDE archive (\u003ca href=\"https://www.ebi.ac.uk/pride/archive/\"\u003ehttps://www.ebi.ac.uk/pride/archive/\u003c/a\u003e) repository. The data files can be accessed with the username\u0026nbsp;\u003cstrong\u003e\u003ca href=\"mailto:[email protected]\" target=\"_blank\"\[email protected]\u003c/a\u003e\u0026nbsp;\u003c/strong\u003eand the password \u003cstrong\u003egtYGw1hi\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMost of the bioinformatics analyses were performed as described previously with some modifications \u003csup\u003e40\u003c/sup\u003e. Theoretical \u003cem\u003ein silico\u003c/em\u003e (2-DE) image of the proteins was acquired by using the tool JVirGel, version 2.0 (\u003ca href=\"http://www.jvirgel.de/index.html\"\u003ehttp://www.jvirgel.de/index.html\u003c/a\u003e) \u003csup\u003e41\u003c/sup\u003e. The subcellular localization of the secretome proteins was predicted using the PSORTb tool, version 3.0.2 (\u003ca href=\"https://www.psort.org/psortb/\"\u003ehttps://www.psort.org/psortb/\u003c/a\u003e) \u003csup\u003e42\u003c/sup\u003e.\u0026nbsp;Signal-peptide bearing proteins were predicted by using the online tool SignalP, version 5.0 (\u003ca href=\"http://www.cbs.dtu.dk/services/SignalP/abstract.php\"\u003ehttp://www.cbs.dtu.dk/services/SignalP/abstract.php\u003c/a\u003e) \u003csup\u003e43\u003c/sup\u003e. Lipoproteins in the secretomes were predicted using\u0026nbsp;the\u0026nbsp;prediction tools\u0026nbsp;LipoP (\u003ca href=\"http://www.cbs.dtu.dk/services/LipoP/\"\u003ehttp://www.cbs.dtu.dk/services/LipoP/\u003c/a\u003e) and PRED-LIPO (\u003ca href=\"http://bioinformatics.biol.uoa.gr/PRED-LIPO/input.jsp\"\u003ehttp://bioinformatics.biol.uoa.gr/PRED-LIPO/input.jsp\u003c/a\u003e) \u003csup\u003e44\u003c/sup\u003e. Further, the\u0026nbsp;prediction tool TatP (\u003ca href=\"http://www.cbs.dtu.dk/services/TatP/\"\u003ehttp://www.cbs.dtu.dk/services/TatP/\u003c/a\u003e), was used to predict proteins secreted via the Twin-arginine translocation pathway (Tat-pathway) \u003csup\u003e45\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunction\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;prediction\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO) of the proteins was analyzed by using the amino acid FASTA sequences. For this, GO annotations were analyzed and plotted using the tools OmicsBox version 1.3.11 (\u003ca href=\"https://www.biobam.com/download-omicsbox/\"\u003ehttps://www.biobam.com/download-omicsbox/\u003c/a\u003e) \u003csup\u003e46\u003c/sup\u003e, and CELLO2GO \u003csup\u003e47\u003c/sup\u003e. Functional association networks within the secretome proteins were determined using the tool STRING (\u003ca href=\"https://string-db.org/\"\u003ehttps://string-db.org/\u003c/a\u003e) \u003csup\u003e48\u003c/sup\u003e. Minimum interaction scores were set at a strong confidence level of 0.7.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of virulence factors in the EVs proteomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVirulentPred (\u003ca href=\"http://203.92.44.117/virulent/\"\u003ehttp://203.92.44.117/virulent/\u003c/a\u003e) was utilized to predict potent virulence factors in the secretomes \u003csup\u003e49\u003c/sup\u003e, along with the Virulence Factor Data Base (VFDB; \u003ca href=\"http://www.mgc.ac.cn/VFs/\"\u003ehttp://www.mgc.ac.cn/VFs/\u003c/a\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStimulation of human whole blood with secretome protein preparations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood collected from a healthy human volunteer was stimulated with \u003cem\u003eP. intermedia\u003c/em\u003e biofilm and planktonic secretome preparations for 24 hours. In a 24-well plate, whole blood was added to the wells and stimulated with 20-\u0026micro;g of secretome protein preparations. After incubating at 37 \u0026deg;C and in 5% CO\u003csub\u003e2\u003c/sub\u003e in air for 24 hours, plasma was separated by centrifugation at 500 \u0026times;g for 3 min and used for cytokine profiling. Duplicate wells containing whole blood but treated with 20 \u0026micro;l sterile PBS were used as negative control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine detection using Proteome Profiler\u003csup\u003eTM\u003c/sup\u003e arrays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma separated from human whole blood stimulated with secretome preparations was applied onto cytokine array membranes for cytokine detection as follows: Nitrocellulose membrane with 36 different capture antibodies spotted in duplicate was used to determine the relative levels of cytokines. Unspecific binding was blocked with assay buffer for 1h at room temperature. The secretome-stimulated PBMC sample (1.5 ml) was diluted in assay buffer with 15 \u0026micro;l of reconstituted human cytokine array detection antibody cocktail and incubated at room temperature for 1h. After washing, the array was treated with streptavidin HRP for 30 min at room temperature on a rocking platform shaker. The array was finally incubated with chemiluminescence reagent for 10 min and images were acquired in Syngene G:Box Imaging System. The positive signals visualized on the array were identified by comparing with the transparency overlay template with the pairs of reference spots in three corners of each array. Mean spot pixel densities were calculated from duplicate spots by analyzing the image using the software provided with G:Box Imaging System.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Kuwait University Grant SRUL 01/14. We thank the Research Administration of Kuwait University for generous funding to the Oral Microbiology Research Laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe authors declare no competing interests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMK: Conceived and designed the study, bioinformatics analyses, performed the experiments, manuscript writing\u003c/p\u003e\n\u003cp\u003eRGB: Performed the experiments, manuscript writing\u003c/p\u003e\n\u003cp\u003eMQ: Data interpretation, manuscript review and writing\u003c/p\u003e\n\u003cp\u003eAA: Data interpretation, manuscript review and writing\u003c/p\u003e\n\u003cp\u003eAE: Data interpretation, manuscript review and writing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the ethical committee of the Health Sciences Center, Kuwait University (DR/EC/3413), and has been carried out in full accordance with the World Medical Association Declaration of Helsinki. The blood donor received written information about the nature and purposes of the study and a written informed consent was obtained upon the volunteer\u0026rsquo;s approval to participate.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Finlay, B. B. \u0026amp; Falkow, S. Common themes in microbial pathogenicity revisited. \u003cem\u003eMicrobiol Mol Biol Rev\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 136-169 (1997).\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\n\u003cp\u003e2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Lee, V. T. \u0026amp; Schneewind, O. 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VirulentPred: a SVM based prediction method for virulent proteins in bacterial pathogens. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 62, doi:10.1186/1471-2105-9-62 (2008). \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Periodontal Pathogen, Planktonic, Secretomes","lastPublishedDoi":"10.21203/rs.3.rs-1078670/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1078670/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003ePrevotella intermedia\u003c/em\u003e is an important species associated with periodontitis. Despite the remarkable clinical significance, little is known about the molecular basis for its virulence. The aim of this study was to characterize the secretome of \u003cem\u003eP. intermedia\u003c/em\u003e in biofilm and planktonic life mode. The biofilm secretome showed 109 proteins while the planktonic secretome showed 136 proteins. The biofilm and the planktonic secretomes contained 17 and 33 signal-peptide bearing proteins, 13 and 18 lipoproteins, respectively. Superoxide reductase, sensor histidine kinase, C40 family peptidase, elongation factor Tu, threonine synthase etc. were unique to biofilm. Of the ~30 proteins with predicted virulence potential from biofilm and planktonic secretomes, only 6 were common between the two groups, implying large differences between biofilm and planktonic modes of \u003cem\u003eP. intermedia\u003c/em\u003e. From gene ontology biofilm secretome displayed a markedly higher percent proteins compared to planktonic secretome in terms of cellular amino acid metabolic process, nitrogen compound metabolic process etc. Inflammatory cytokine profile analysis revealed that only the biofilm secretome, not the planktonic one, induced important cytokines such as MIP-1a/MIP-1b, IL-1b, and IL-8. In conclusion, the revealed differences in the protein profiles of \u003cem\u003eP. intermedia\u003c/em\u003e biofilm and planktonic secretomes may trigger further questions about molecular mechanisms how this species exerts its virulence potential in the oral cavity.\u003c/p\u003e","manuscriptTitle":"Proteomic Analysis of the Periodontal Pathogen Prevotella Intermedia Secretomes in Biofilm and Planktonic Lifestyles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-12-06 17:10:07","doi":"10.21203/rs.3.rs-1078670/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-01-27T08:50:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-01-25T12:31:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-12-09T10:54:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"346a2bdf-8ab2-498f-aec2-c770c2c88981","date":"2021-12-03T08:28:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-12-03T08:22:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-12-03T08:10:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-12-03T06:47:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-12-03T06:19:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-11-14T09:40:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c4c2be03-a1f8-4b3b-aa7f-317dce1d7695","owner":[],"postedDate":"December 6th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":8955209,"name":"General Microbiology"}],"tags":[],"updatedAt":"2022-04-04T18:44:27+00:00","versionOfRecord":{"articleIdentity":"rs-1078670","link":"https://doi.org/10.1038/s41598-022-09085-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2022-04-04 18:44:27","publishedOnDateReadable":"April 4th, 2022"},"versionCreatedAt":"2021-12-06 17:10:07","video":"","vorDoi":"10.1038/s41598-022-09085-0","vorDoiUrl":"https://doi.org/10.1038/s41598-022-09085-0","workflowStages":[]},"version":"v1","identity":"rs-1078670","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1078670","identity":"rs-1078670","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}