PMC:6599329 / 5408-14890
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6599329","sourcedb":"PMC","sourceid":"6599329","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6599329","text":"Results\n\nBiofilm forming potential of E. faecalis SK460\nConfocal Laser Scanning Microscopy analysis evidenced the high biofilm forming ability of E. faecalis SK460 (Fig. 1). The 24 h old biofilm showed an average thickness of approximately 40 μm.\nFig. 1 Confocal laser scanning microscopy images of biofilm formation of E. faecalis SK460 at 12 h and 24 h. The images are processed using NIS-Element AR software, version 4.00.04\n\nProteome profile obtained in planktonic and biofilm stages\nLabel-free quantitative proteomics identified 657 proteins from planktonic stages and 553 proteins from biofilm stages. Of these, 233 (29.6%) and 129 (16.4%) proteins were identified exclusively in the planktonic and biofilm stages respectively and 424 (53.9%) proteins were detected at both stages.\n\nPhysico-chemical properties of identified proteome\nThe hydrophobic nature of the identified proteins was calculated using the GRAVY (grand average hydropathy) tool and the score obtained ranges between − 1.6 and 1 (Additional file 2: Figure S1a). Nearly 88% of the proteins were hydrophilic (\u003c 0) and the rest were hydrophobic or membranous (\u003e 0) in nature. The compute pI/MW tool revealed that the extracted proteins were within the pI range of 3.5–11.5 and molecular weight of 4–186 kDa (Additional file 3: Figure S1b). 90% of the proteins were within the range of 80 kDa.\n\nFunctional categorization of proteome at planktonic and biofilm stages\n\nKEGG pathways\nThe proteins identified in the planktonic and biofilm stages were assigned to define KEGG pathways. The major pathways were associated with ribosome (72 hits), pyruvate metabolism (27 hits), pyrimidine metabolism (50 hits), amino acid biosynthesis (69 hits) and antibiotics (122 hits), Fatty acid metabolism (15 hits), RNA degradation (13 hits), biosynthesis of secondary metabolites (157 hits), glycolysis/gluconeogenesis (40 hits), citrate cycle (TCA cycle) (7 hits), microbial metabolism in diverse environments (110 hits), propanoate metabolism (11 hits) and butanoate metabolism (9 hits). Furthermore, the proteins upregulated/unique to biofilm stages were assigned to KEGG pathways and revealed the enrichment of glycolysis/gluconeogenesis, pyruvate metabolism, biosynthesis of secondary metabolites, microbial metabolism in diverse environments and arginine metabolism (Fig. 2). More than 50% of the total proteins involved in these pathways were detected to be upregulated in biofilm stages of SK460.\nFig. 2 KEGG pathways and molecular functions assigned to categorize the upregulated proteins obtained in biofilm stages of E. faecalis SK460\n\nBiological processes\nThe proteins were clustered based on gene ontology (GO) analysis and its relative expression at planktonic and biofilm stages were analyzed. Comparative proteome analysis revealed that several ribosomal proteins and proteins involved in DNA replication and transcription were found to be normally expressed or slightly upregulated. The relative abundance of some proteins involved in nucleotide metabolism and protein biosynthesis was affected to differing degrees under biofilm conditions. Major functional categories of proteins upregulated in biofilm stages are listed in Table 1.\nTable 1 List of proteins enhanced in biofilm stages of E. faecalis SK460\nNCBI-ID Pathways/cellular processes\nAmino acid metabolism\n NP_816782.1 Succinyl-diaminopimelate desuccinylase (EF_3178)\n NP_816328.1 MTA/SAH nucleosidase (pfs)\n NP_815300.1 Cysteine synthase A (cysK)\n NP_815281.1 Chorismate synthase (aroC)\n NP_815821.1 Glutamine synthetase, type I (glnA)\n NP_814529.1 S-adenosylmethionine synthetase (metK)\n NP_815283.1 3-phosphoshikimate 1-carboxyvinyltransferase (aroA)\n NP_816193.1 Serine hydroxymethyltransferase (glyA)\n NP_816069.1 Threonine synthase (thrC)\n NP_815279.1 3-deoxy-7-phosphoheptulonate synthase (EF_1562)\n NP_816700.1 Bifunctional glutamate--cysteine ligase/glutathione synthetase (gshF)\n NP_816650.1 Glutamyl-aminopeptidase (pepA)\n NP_814940.1 Acetolactate synthase (EF_1213)\n NP_815294.1 Dihydrofolate reductase (folA)\nGlycolysis, citric acid cycle, pentose phosphate pathway\n NP_815638.1 Triosephosphate isomerase (tpiA)\n NP_815639.1 Phosphoglycerate kinase (pgk)\n NP_814897.1 Fructose-bisphosphate aldolase (fba)\n NP_813994.1 Phosphoglycerate mutase (gpmA)\n NP_815245.1 Glyceraldehyde 3-phosphate dehydrogenase (gap-1)\n NP_815637.1 Enolase (eno)\n NP_815137.1 Glucose-6-phosphate isomerase (pgi)\n NP_815075.1 Pyruvate dehydrogenase complex, E1 component subunit beta (pdhB)\n NP_815076.1 Dihydrolipoamide acetyltransferase (aceF)\n NP_816104.1 Pyruvate carboxylase (pycA)\n NP_813976.1 Deoxyribose-phosphate aldolase (deoC)\n NP_813986.1 Phosphopentomutase (deoB)\n NP_814782.1 6-phosphogluconate dehydrogenase (gnd)\nStress response, chaperones and DNA repair\n NP_814237.1 OsmC/Ohr family protein (EF_0453)\n NP_816831.1 Dps family protein (EF_3233)\n NP_816649.1 Thioredoxin family protein (EF_3036)\n NP_814938.1 NADH peroxidase (npr)\n NP_815447.1 General stress protein (EF_1744)\n NP_816369.1 Alkyl hydroperoxide reductase subunit C (ahpC)\n NP_813885.1 gls24 protein\n NP_816648.1 Universal stress protein (EF_3035)\n NP_816867.1 Glutathione reductase (gor)\n NP_816700.1 Bifunctional glutamate--cysteine ligase/glutathione synthetase (gshF)\n NP_816368.1 Thioredoxin reductase/glutathione-like protein (EF_2738)\n NP_814049.1 L-lactate dehydrogenase (ldh1)\n NP_816550.1 AhpC/TSA family protein (EF_2932)\n NP_815246.2 GTPase ObgE\n NP_815059.1 Thioredoxin reductase (trxB)\n NP_815353.1 Transcriptional repressor CodY (codY)\n NP_816010.1 ATP-dependent Clp protease, ATP-binding protein ClpB (clpB)\n NP_814518.1 ATP-dependent Clp protease proteolytic subunit (clpP)\n NP_816516.1 Cyclophilin type peptidyl-prolyl cis-trans isomerase (EF_2898)\n NP_814457.1 ATP-dependent Clp protease, ATP-binding protein ClpE (clpE)\n NP_816532.1 Transcription elongation factor GreA\n NP_815030.1 dnak protein\n NP_815032.1 dnaJ protein\n NP_815029.1 Heat shock protein GrpE (grpE)\n NP_814465.1 Trigger factor (TIG)\n NP_816273.1 GroS (10 kDa chaperonin)\n NP_816272.1 GroL(60 kDa chaperonin)\n NP_814060.1 Fn1610 (33 kDa chaperonin) (hslO)\n NP_814686.1 Uracil-DNA glycosylase (ung)\n NP_814720.1 DNA repair protein RecN (recN)\nRhamnose biosynthesis\n NP_815854.1 dTDP-glucose 4,6-dehydratase (rfbB)\n NP_815853.1 dTDP-4-dehydrorhamnose reductase (EF_2191)\n NP_815856.1 Glucose-1-phosphate thymidylyltransferase (rfbA)\nArginine metabolism\n NP_813908.1 Ornithine carbamoyltransferase (arcB)\n NP_815423.1 Carbamoyl phosphate synthase small subunit (carA)\n NP_814373.1 Ornithine cyclodeaminase (EF_0616)\n NP_813909.1 Carbamate kinase (arcC1)\n NP_813907.1 Arginine deiminase (arcA)\n NP_814719.1 ArgR family transcriptional regulator (EF_0983)\nQuorum sensing and pheromone associated lipoprotein\n NP_814911.1 S-ribosylhomocysteinase (luxS)\n NP_816853.1 Pheromone cAD1 lipoprotein (EF_3256)\n NP_814793.1 Pheromone binding protein (EF_1060)\n NP_815061.1 Pheromone cAM373 lipoprotein (EF_1340)\nAdhesion associated proteins\n NP_814266.1 Aggregation substance (EF_0485)\n NP_814342.1 Adhesion lipoprotein (EF_0577)\n NP_814975.1 Fibronectin/fibrinogen binding protein (EF_1249)\nAutolysin and D-Alanylation of teichoic acid\n NP_814543.1 Autolysin (EF_0799)\n NP_816376.1 D-alanine--poly(phosphoribitol) ligase subunit 2 (dltC)\n NP_816375.1 dltD protein (dltD)\nIn general, major glycolytic enzymes and several enzymes in the citric acid cycle and pentose phosphate pathway were found to be in higher abundance in biofilm stages of SK460. Enzymes involved in rhamnose biosynthesis which forms the part of enterococcal polysaccharide antigen gene cluster was also found to be upregulated in biofilm stages. Compared to planktonic forms, Enterococcal biofilms showed a hike in stress response factors, DNA repair system, molecular chaperones, pheromone associated lipoproteins and other adhesion associated proteins. Besides, the present proteome analysis established the role of luxS mediated quorum sensing in enterococcal biofilm development.\nSTRING analysis predicted the molecular interactions among the highly expressed proteins in biofilm stages with enrichment p-value \u003c 1.0e-16 and is shown in Fig. 3. The functional interactome of the whole upregulated protein sets is depicted in Additional file 2: Figure S2.\nFig. 3 STRING analysis (Version 10.5) showing the predicted molecular action among the upregulated proteins in biofilm stages. The gene nomenclatures are depicted in Table 1. Colored nodes: query proteins and first shell of interactors. Empty nodes: proteins of unknown 3D structure. Filled nodes: 3D structure is known/predicted. Edges represent protein-protein associations\n\nValidation of selected biofilm associated genes by RT-PCR\nThe genes rfbB (Rhamnose biosynthesis), arcA (Arginine deiminase), luxS (Quorum sensing), cAD1 (Pheromone cAD1 lipoprotein) and fbn (Fibrinogen/Fibronectin binding protein associated with adhesion) were selected to monitor the fold change expression in biofilm stage when compared to planktonic stage and the results are shown in Fig. 4. All these five genes were found to be upregulated and adhesion protein (fbn) showed a comparatively large fold increase in expression. This substantiates the significant role of these pathways in the development of mature biofilm.\nFig. 4 Real time qPCR validation of selected biofilm associated genes. Fold change expression of genes observed in biofilm stage with respect to its corresponding planktonic 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