PMC:6599329 / 14892-27593 JSONTXT

{"project":"2_test","denotations":[{"id":"31253082-29358339-12221358","span":{"begin":2290,"end":2291},"obj":"29358339"},{"id":"31253082-15947432-12221359","span":{"begin":2730,"end":2732},"obj":"15947432"},{"id":"31253082-28285095-12221359","span":{"begin":2730,"end":2732},"obj":"28285095"},{"id":"31253082-11807075-12221360","span":{"begin":3296,"end":3298},"obj":"11807075"},{"id":"31253082-19253936-12221361","span":{"begin":3300,"end":3302},"obj":"19253936"},{"id":"31253082-12655459-12221362","span":{"begin":3380,"end":3382},"obj":"12655459"},{"id":"31253082-17636987-12221363","span":{"begin":3669,"end":3671},"obj":"17636987"},{"id":"31253082-11375188-12221364","span":{"begin":3832,"end":3834},"obj":"11375188"},{"id":"31253082-17020544-12221365","span":{"begin":4573,"end":4575},"obj":"17020544"},{"id":"31253082-21761944-12221366","span":{"begin":4577,"end":4579},"obj":"21761944"},{"id":"31253082-22096200-12221367","span":{"begin":4823,"end":4825},"obj":"22096200"},{"id":"31253082-15633107-12221368","span":{"begin":5785,"end":5787},"obj":"15633107"},{"id":"31253082-24035282-12221369","span":{"begin":6353,"end":6355},"obj":"24035282"},{"id":"31253082-25403660-12221369","span":{"begin":6353,"end":6355},"obj":"25403660"},{"id":"31253082-30271935-12221369","span":{"begin":6353,"end":6355},"obj":"30271935"},{"id":"31253082-9712783-12221370","span":{"begin":6806,"end":6808},"obj":"9712783"},{"id":"31253082-19581393-12221371","span":{"begin":6810,"end":6812},"obj":"19581393"},{"id":"31253082-25035517-12221372","span":{"begin":6843,"end":6845},"obj":"25035517"},{"id":"31253082-24755907-12221373","span":{"begin":7005,"end":7007},"obj":"24755907"},{"id":"31253082-14987774-12221374","span":{"begin":7297,"end":7299},"obj":"14987774"},{"id":"31253082-24727224-12221375","span":{"begin":8001,"end":8003},"obj":"24727224"},{"id":"31253082-27080421-12221376","span":{"begin":8627,"end":8629},"obj":"27080421"},{"id":"31253082-16897563-12221377","span":{"begin":8776,"end":8778},"obj":"16897563"},{"id":"31253082-15870324-12221378","span":{"begin":8780,"end":8782},"obj":"15870324"},{"id":"31253082-25249282-12221379","span":{"begin":9178,"end":9180},"obj":"25249282"},{"id":"31253082-21209892-12221380","span":{"begin":9536,"end":9537},"obj":"21209892"},{"id":"31253082-25431047-12221381","span":{"begin":9539,"end":9541},"obj":"25431047"},{"id":"31253082-21903750-12221382","span":{"begin":9666,"end":9668},"obj":"21903750"},{"id":"31253082-22782954-12221383","span":{"begin":9963,"end":9965},"obj":"22782954"},{"id":"31253082-26351286-12221384","span":{"begin":9993,"end":9995},"obj":"26351286"},{"id":"31253082-24628034-12221385","span":{"begin":10170,"end":10172},"obj":"24628034"},{"id":"31253082-19528211-12221386","span":{"begin":10965,"end":10966},"obj":"19528211"},{"id":"31253082-16790791-12221387","span":{"begin":11620,"end":11621},"obj":"16790791"}],"text":"Discussion\nE. faecalis is a clinically relevant hospital associated pathogen and the recalcitrant enterococcal biofilms are known to play a foremost role in establishing chronic infections. The wound isolate E. faecalis SK460 was proved to be a robust biofilm former by confocal laser scanning microscopy. Though SK460 is a potent biofilm former, it is devoid of several well-known biofilm determinants including fsr quorum signaling system and gelatinase production. Previous studies have established the role of fsr regulated gelatinase production and enterococcal surface protein in Enterococcal biofilm development. Besides these aforementioned determinants, several other factors are yet to be elucidated to pick out the unidentified factors associated with enterococcal biofilm. Hence, our prime focus was to decipher the metabolic pathways and physiological processes related to the Enterococcal biofilm development utilizing the label-free quantitative proteomic approach.\nIn the present investigation, the majority of proteins involved in basic survival mechanisms including DNA replication and transcription were either normally expressed or slightly upregulated in biofilm stages. Starting from the planktonic form to microcolonies and maturing biofilm, an intracellular pool of carbohydrates, amino acids and lipids is required to facilitate the development of biomass required for the sessile growth period. Besides these, certain putative transcriptional regulators and DNA binding response regulators have also shown an increasing trend in biofilm stages. The differential expression of major metabolic pathways and cellular processes upregulated in biofilm stages are discussed in this section.\n\nPathways upregulated in biofilm stages\n\nAmino acid metabolism\nIn the present investigation, various enzymes including chorismate synthase (3.97 fold), 3-phosphoshikimate 1-carboxyvinyltransferase (1.5 fold), and 3-deoxy-7-phosphoheptulonate synthase (1.56 fold) which are involved in the biosynthesis of aromatic amino acids were found to be upregulated in biofilm stages. A similar trend of enhanced expression of aromatic amino acid biosynthesis enzymes has already been reported in a previous proteomic study performed on strong and weak biofilm forming E. faecalis strains [8]. Other major amino acid biosynthesis enzymes upregulated were Succinyl-diaminopimelate desuccinylase (6.35 fold), MTA/SAH nucleosidase (2.88 fold), cysteine synthase A (3.387 fold), Glutamine synthetase (5.81 fold), Acetolactate synthase (1.89 fold), etc. Previous studies on several other bacterial genera also revealed the role of amino acids in the formation of tight microcolonies and in maintaining the stability of mature biofilm [10–12]. Hence it is evident that during the biofilm maturation process, a series of amino acids and its related metabolites will be upregulated for contributing to extracellular matrix component of robust biofilms.\n\nCarbohydrate metabolism\nThe present study evidenced the augmented production of seven major glycolytic enzymes (2.5 to 9 fold change) in biofilm stages compared to planktonic forms as listed in Table 1. Previous studies have shown the significant role of Glyceraldehyde 3-phosphate dehydrogenase in P. aeruginosa and Staphylococcus xylosus biofilms [13, 14] and also an upregulated expression in microaerophilic conditions in E.coli [15]. It is assumed that the cells within the biofilm will be in microaerophilic condition and needs a high level expression of these enzymes for adapting in the oxygen-limited environment. Another glycolytic enzyme phosphoglycerate mutase was found to be upregulated in S.xylosus biofilm [16] and is said to have a bifunctional role which helps in the synthesis of various EPSs, thus playing a significant role in the synthesis of core biofilm matrix [17]. Thus the glycolytic pathway is found to be one of the essential factors among biofilm survival mechanisms. Pyruvate dehydrogenase complex and several enzymes involved in Tricarboxylic acid (TCA) cycle and pentose phosphate pathways were also observed to be enhanced during the biofilm formation of SK460.\n\nStress response factors and protein folding\nSeveral proteins involved in stress response including universal stress protein (10.69 fold), and General stress protein (3.42 fold) were identified to be steadily increased in biofilm stages. Similarly, universal stress proteins were found to be upregulated in Porphyromonas gingivalis and Sulfolobus solfataricus biofilms and showed impaired biofilm upon inactivation of these genes [18, 19]. Signaling pathways for inducing stress response within the biofilm are cell-density dependent quorum sensing and the starvation–activated stringent response where the latter helps bacteria in adapting to nutrient deprivation. Nguyen et al. [20] identified the role of this stringent response in protecting bacteria within the biofilm from antimicrobial stress. It also activates the production of catalase and superoxide dismutase in stress condition. Enhanced expression of GTPase ObgE (1.53 fold) suggested the tendency of biofilm cells to attain the persistence nature in response to nutrient deprivation as suggested by previous findings [21]. Similarly, proteins like DPS family protein (5.41 fold), Thioredoxin family protein (4.75 fold), Alkyl hydroperoxide reductase (2.33 fold) and H2O2 scavenging proteins (9.11 fold) involved in reactive oxygen stress were also found to be over synthesized. It was also noticed that Gls24 is overexpressed by 9.2 fold in biofilm cells, suggesting its role in Enterococcal biofilm which is in accordance with the previous reports. Gls24 was found to be related to the stress and virulence of E. faecalis and was considered as a possible immunotherapy target [22].\nChaperonins and chaperones including GroL (1.63 fold), GroS (3.49 fold), several heat shock proteins, etc. were also found to be relatively abundant in SK460 biofilm. Enhanced levels of chaperones ensure the proper protein folding which enables E. faecalis to thrive within the biofilm. Clp proteases involved in the degradation of misfolded proteins also showed elevated expression in biofilm stages. Several studies have proved the role of chaperone DnaK in curli-dependent biofilm formation and are considered as a potential target for anti-biofilm compounds [23–25].\n\nRhamnose biosynthesis\nRhamnose biosynthesis enzymes including glucose-1-phosphate thymidylyltransferase (RfbA), dTDP-glucose 4,6-dehydratase (RfbB) and dTDP-4-dehydrorhamnose reductase (RfbD) were found to be more than threefold enhanced expression in the biofilm stage. Rhamnose biosynthesis enzymes belong to Enterococcal polysaccharide antigen (epa) gene cluster and were proved to have a major role in virulence in mouse peritonitis model [26, 27] and intestinal colonization [28]. This rhamnopolysaccharide epa also confers protection against high salt concentration and are said to be involved in osmotic stress response in E. faecalis [29]. Epa also confers resistance to various stresses including those of oxidative stress, stress towards ethanol, bile acids, detergent SDS and antimicrobial peptides. Decreased EPS production and colonization was observed due to knockout of rhamnose biosynthesis in Azospirillum brasilense [30]. Thus rhamnopolysaccharide serves as a major extracellular matrix component as well as enables the cells to survive within the stressed environment of the matrix.\n\nArginine metabolism\nArginine deiminase (ArcA), ornithine carbamoyltransferase (ArcB) and carbamate kinase (ArcC) are enzymes involved in arginine deiminase (ADI) pathway and were found to be highly expressed (ranging from 1.9 to 8 fold) in E. faecalis biofilm in the present study. Ammonia synthesis via the ADI pathway is important to reduce the pH stress within the microcolonies. Mutants of arginine deiminase have showed decreased viability in S. epidermidis biofilm, and ADI is proved to have a role in pH homeostasis in biofilms [31]. Transcription of arc operon is often arginine dependent via ArgR family of regulators (ArgR and AhrC) which were found to be active in biofilm stages. ADI pathway is generally induced under anaerobic conditions and hence this may have a role in the oxygen reduced environment within the biofilm.\n\nQuorum sensing (QS) and pheromone associated proteins\nS-ribosylhomocysteinase (LuxS) was shown to have a rising trend with more than threefold increased expression in biofilm stages. Previous studies evidenced that luxS mutants developed an altered biofilm and exhibited enhanced cell-surface hydrophobicity in E. faecalis [32]. Several other studies also had a similar observation of the negative impact of luxS mutants in Streptococcus anginosus, and S. mutans biofilms [33, 34]. But still, the role of luxS dependent quorum sensing is not well established in E. faecalis biofilm.\nPheromone cAD1 lipoprotein (1.91 fold) and other pheromone associated proteins were upregulated in biofilm stages. Surface-associated pheromone binding lipoproteins act as essential components for adhesion, colonization and virulence and are also involved in stress response. Varahan et al. [35] showed that pheromone transporter mutants displayed altered biofilm architecture with a significant reduction in biofilm biomass compared to wild type suggesting the role of pheromones and associated proteins in biofilm development.\n\nAdhesion associated proteins\nAggregation substance was already proved to have a role in Enterococcal biofilm formation [3, 36]. Adhesion lipoproteins are surface-associated lipoproteins involved in Enterococcal adherence, colonization and virulence [37] and are directly linked to biofilm formation as in agreement with our findings. Fibronectin/fibrinogen binding proteins also showed more than tenfold increase in biofilm stages and were suggested to have a significant role in E. faecalis associated urinary tract infections in a murine model [38] and endocarditis in rats [39]. Fibronectin binding protein mutants of community-associated MRSA losses the ability for fibronectin binding thereby preventing the establishment of biofilm in host tissue [40]. These proteins were assumed to be associated with bacterial aggregation and involve in primary attachment to a surface and hence can be considered as a suitable biofilm inhibiting target.\n\nOther major factors\nSeveral DNA binding response regulators, phosphotransferase system transporter proteins, Autolysin, D-alanylation of lipoteichoic acid (dltABCD), etc. were also found to be more than two-fold upregulated in biofilm stages. Of these, autolysin is well known to be associated with the eDNA release in the matrix and structural integrity of the biofilm. Previous report showed that autolysin deficient mutants had defects in primary attachment and eDNA release which are mainly associated with the accumulative phase for maturation and structural stability of biofilm in E. faecalis [5]. These mutants lack DNase1-sensitive fibrous network which is found to have a role in biofilm stability. D-alanylation of lipoteichoic acid (dltC and dltD) was also found to be abundant in biofilm mode (2.36 to 4.17 fold), possibly incorporating lipoteichoic acid into the EPS matrix. Lack of d-alanine esters results in a stronger negative net charge on the bacterial cell surface and dlt mutants showed a reduction in biofilm formation on polystyrene surfaces. Furthermore, dltABCD operon is involved in the pathogenesis of E. faecalis leading to enhanced biofilm formation, host tissue attachment and increased resistance to antimicrobial peptides [6].\nSTRING analysis revealed that upregulated genes including stress response factors, major glycolytic enzymes, arginine metabolism and rhamnose biosynthesis were either directly or indirectly poses a close molecular interaction (p-value \u003c 1.0e-16), thereby regulating each other in stressful environment prevailing during biofilm development. Among the upregulated cellular processes, rfbB (Rhamnose biosynthesis), arcA (Arginine deiminase), luxS (Quorum sensing), cAD1 (Pheromone cAD1 lipoprotein) and fbn (Fibrinogen/Fibronectin binding protein associated with adhesion) were selected for gene expression analysis and were found to be enhanced by 2.29, 4.03, 2.35, 1.5 and 13.87 fold respectively in biofilm stages. Of these, luxS mediated quorum sensing system is attributed to play a major role in E. faecalis SK460 biofilm which is devoid of fsr two-component signal transduction system. Accordance of the proteome data with RT-PCR results confirms the reliability of the analysis, serving as a validation for the identified determinants of Enterococcal biofilm development."}