Comparative proteomic analysis of Streptococcus suis biofilms and planktonic cells that identified biofilm infection-related immunogenic proteins

Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections.     

Biofilm Formation of Streptococcus equi ssp. zooepidemicus and Comparative Proteomic Analysis of Biofilm and Planktonic Cells

Streptococcus equi ssp. zooepidemicus (SEZ) is responsible for a wide variety of infections in many species, including pigs, horses and humans. Biofilm formation is essential for pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. However, the ability of SEZ to form biofilms is unclear. Furthermore, the mechanisms underlying SEZ biofilm formation and their attributes are poorly understood. In this study, scanning electron microscopy (SEM) demonstrated that SEZ strain ATCC35246 formed biofilms comprising a thick, heterogeneous layer with clumps on the coverslips when incubated for 24 h. In addition, we used a two-dimensional gel electrophoresis (2-DE) based approach to characterize differentially expressed protein in SEZ biofilms compared with their planktonic counterparts. The results revealed the existence of 24 protein spots of varying intensities, 13 of which were upregulated and 11 were downregulated in the SEZ biofilm compared with the planktonic controls. Most of proteins expressed during biofilm formation were associated with metabolism, adhesion, and stress conditions. These observations contribute to our understanding of the SEZ biofilm lifestyle, which may lead to more effective measures to control persistent SEZ infections.     

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

Proteomic analysis of cytoplasmic and surface proteins from yeast cells,hyphae, and biofilms of Candida albicans

Candida albicans is a human commensal and opportunistic pathogen that participates in biofilm formation on host surfaces and on medical devices. We used DIGE analysis to assess the cytoplasmic and non‐covalently attached cell‐surface proteins in biofilm formed on polymethylmethacrylate and planktonic yeast cells and hyphae. Of the 1490 proteins spots from cytoplasmic and 580 protein spots from the surface extracts analyzed, 265 and 108 were differentially abundant respectively (> 1.5‐fold, p <0.05). Differences of both greater and lesser abundance were found between biofilms and both planktonic conditions as well as between yeast cells and hyphae. The identity of 114 cytoplasmic and 80 surface protein spots determined represented 73 and 25 unique proteins, respectively. Analyses showed that yeast cells differed most in cytoplasmic profiling while biofilms differed most in surface profiling. Several processes and functions were significantly affected by the differentially abundant cytoplasmic proteins. Particularly noted were many of the enzymes of respiratory and fermentative pentose and glucose metabolism, folate interconversions and proteins associated with oxidative and stress response functions, host response, and multi‐organism interaction. The differential abundance of cytoplasmic and surface proteins demonstrated that sessile and planktonic organisms have a unique profile.     

Proteomics for the analysis of the Candida albicans biofilm lifestyle

Candida albicans is an opportunistic pathogenic fungus capable of causing infections in immunocompromised patients. Candidiasis is often associated with the formation of biofilms on the surface of inert or biological materials. Biofilms are structured microbial communities attached to a surface and encased within a matrix of exopolymeric substance (EPS). At present, very little is known about the changes in protein profiles that occur during the transition from the planktonic to the biofilm mode of growth. Here, we report the use of proteomics for the comparative analysis of subcellular fractions obtained from C. albicans biofilm and planktonic cultures, including cell surface-associated proteins and secreted components present in liquid culture supernatants (for planktonic cultures) and EPS (for biofilms). The analysis revealed a high degree of similarity between the protein profiles associated with the planktonic and biofilm extracts, and led to the identification of several differentially expressed protein spots. Among the differentially expressed proteins, there was a preponderance of metabolic enzymes that have been described as cell surface proteins and immunodominant antigens. Proteins found in the biofilm matrix included a few predicted to form part of the secretome, and also many secretion-signal-less proteins. These observations contribute to our understanding of the C. albicans biofilm lifestyle.     

Candida albicans biofilm formation is associated with increased anti-oxidative capacities

Candida albicans is a common, opportunistic, human fungal pathogen that causes a variety of mucosal and systemic afflictions. It exists in nature both in the biofilm or the sessile phase, as well as in the free-floating or the planktonic phase. Candida biofilms, in particular, display unique characteristics that confer survival advantages over their planktonic counterparts, such as their recalcitrance to common antifungals. The mechanisms underlying Candida biofilm formation and their attributes are poorly understood. In this study, we used a 2-DE-based approach to characterize the protein markers that are differentially expressed in Candida biofilms in comparison to their planktonic counterparts. Using tandem mass spectrometric analysis, we have identified a significant number of proteins including alkyl hydroperoxide reductase, thioredoxin peroxidase, and thioredoxin involved in oxidative stress defenses that are upregulated in the biofilm phase. These proteomic findings were further confirmed by real-time PCR and lucigenin-based chemiluminescence assays. In addition, we demonstrate that a drug target for the new antifungal agent echinocandin, is abundantly expressed and significantly upregulated in Candida biofilms. Taken together, these data imply that the biofilm mode, Candida, compared with their planktonic counterparts, exhibits traits that can sustain oxidative stress (anti-oxidants), and thereby exert resistance to commonly used antifungals.     

Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200

This study investigated the relative abundance of extracellular and cell wall associated proteins (exoproteome), cytoplasmic proteins (proteome), and related phenotypic traits of Lactobacillus plantarum grown under planktonic and biofilm conditions. Lactobacillus plantarum DB200 was preliminarily selected due to its ability to form biofilms and to adhere to Caco2 cells. As shown by fluorescence microscope analysis, biofilm cells became longer and autoaggregated at higher levels than planktonic cells. The molar ratio between glucose consumed and lactate synthesised was markedly decreased under biofilm compared to planktonic conditions. DIGE analysis showed a differential exoproteome (115 protein spots) and proteome (44) between planktonic and biofilm L. plantarum DB200 cells. Proteins up‐ or downregulated by at least twofold (p < 0.05) were found to belong mainly to the following functional categories: cell wall and catabolic process, cell cycle and adhesion, transport, glycolysis and carbohydrate metabolism, exopolysaccharide metabolism, amino acid and protein metabolisms, fatty acid and lipid biosynthesis, purine and nucleotide metabolism, stress response, oxidation/reduction process, and energy metabolism. Many of the above proteins showed moonlighting behavior. In accordance with the high expression levels of stress proteins (e.g., DnaK, GroEL, ClpP, GroES, and catalase), biofilm cells demonstrated enhanced survival under conditions of environmental stress.     

Factors Affecting Catalase Expression in Pseudomonas aeruginosa Biofilms and Planktonic Cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 μM as FeCl₃) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

Impact of rpoS deletion on the proteome of Escherichia coli grown planktonically and as biofilm

To investigate the role of rpoS in gene expression of Escherichia coli cells grown as biofilms, we compared the proteomes of a rpoS mutant and the wild-type strain. Experiments were performed on planktonic cells (in exponential or stationary growth phase) and biofilms developed on glass wool. Spot-by-spot comparison of gels obtained from biofilm and planktonic wild-type organisms showed that the intensity of between 22 and 30% of detected spots was affected by the growth mode, depending of the control used. Principal component analysis, used to interpret the variations in protein spot densities, discriminated exponential-phase cells (wild-type and mutant) from the other incubation conditions and secondarily 72-old cultures. The statistical analysis demonstrated that the rpoS mutation did not significantly modify the proteome of exponential-growth phase cells, the differences involving only 3% of the proteome. However, increasing the incubation time from 8 to 72 h noticeably increased the number of changed proteins. A cluster analysis showed that RpoS plays a role in the special nature of the gene expression of biofilm cells but lower than in stationary-phase bacteria. We identified 35 rpoS-regulated proteins that were already or not described as controlled by this sigma factor. For some of them, the mode of regulation by RpoS was obviously dependent on the culture condition (planktonic vs biofilm).     

Comparative proteomic analysis of biofilm and planktonic cells of Neisseria meningitidis

Neisseria meningitidis is a commensal of the human nasopharynx occasionally causing invasive disease. In vitro biofilms have been employed to model meningococcal carriage. A proteomic analysis of meningococcal biofilms was conducted and metabolic changes related to oxygen and nutrient limitation and upregulation of proteins involved in ROS defense were observed. The upregulated MntC which protects against ROS was shown to be required for meningococcal biofilm formation, but not for planktonic growth. ROS-induced proteomic changes might train the biofilm to cope with immune effectors.     

Persister cells in a biofilm treated with a biocide

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.     

Characterization of Phenotypic Changes in Pseudomonas putida in Response to Surface-Associated Growth

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface. A switch between planktonic and sessile growth is believed to result in a phenotypic change in bacteria. In this study, a global analysis of physiological changes of the plant saprophyte Pseudomonas putida following 6 h of attachment to a silicone surface was carried out by analysis of protein profiles and by mRNA expression patterns. Two-dimensional (2-D) gel electrophoresis revealed 15 proteins that were up-regulated following bacterial adhesion and 30 proteins that were down-regulated. N-terminal sequence analyses of 11 of the down-regulated proteins identified a protein with homology to the ABC transporter, PotF; an outer membrane lipoprotein, NlpD; and five proteins that were homologous to proteins involved in amino acid metabolism. cDNA subtractive hybridization revealed 40 genes that were differentially expressed following initial attachment of P. putida. Twenty-eight of these genes had known homologs. As with the 2-D gel analysis, NlpD and genes involved in amino acid metabolism were identified by subtractive hybridization and found to be down-regulated following surface-associated growth. The gene for PotB was up-regulated, suggesting differential expression of ABC transporters following attachment to this surface. Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes involved in polysaccharide biosynthesis. Immunoblot analysis of PilA and FliC confirmed the presence of flagella in planktonic cultures but not in 12- or 24-h biofilms. In contrast, PilA was observed in 12-h biofilms but not in planktonic culture. Recent evidence suggests that quorum sensing by bacterial homoserine lactones (HSLs) may play a regulatory role in biofilm development. To determine if similar protein profiles occurred during quorum sensing and during early biofilm formation, HSLs extracted from P. putida and pure C(12)-HSL were added to 6-h planktonic cultures of P. putida, and cell extracts were analyzed by 2-D gel profiles. Differential expression of 16 proteins was observed following addition of HSLs. One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition. The other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth. The results presented here demonstrate that P. putida undergoes a global change in gene expression following initial attachment to a surface. Quorum sensing may play a role in the initial attachment process, but other sensory processes must also be involved in these phenotypic changes.     

Oral anaerobes cannot survive oxygen stress without interacting with facuItative/aerobic species as a microbial commmunity

D.J. Bradshaw, P.D. Marsh, G.K. Watson and C. Allison. 1997. Anaerobic bacteria are found commonly as components of mixed culture biofilms in many aerated habitats, including the mouth. Previous studies showed that anaerobes could survive in planktonic and biofilm communities in aerated conditions when part of a community including facultative and/or aerobic species, and the numbers and proportions of anaerobic species increased as biofilms aged. When the obligate anaerobes were grown in the absence of aerobic/facultative species, however, they were unable to grow in either the planktonic or biofilm culture. The mean survival times of organisms in the aerated culture containing four anaerobic species varied from around 5 min for Fusobacterium nucleatum and Veillonella dispar , to less than 4 min for Porphyromonas gingivalis and Prevotella nigrescens . In addition, in this culture, the biofilm mode of growth did not provide a haven for these bacteria in the absence of oxygen-consuming species.     

A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode

Escherichia coli 0157:H7 biofilms were studied by a new method of cultivation in order to identify some of the proteins involved in the biofilm phenotype. A proteomic analysis of sessile or planktonic bacteria of the same age was carried out by two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Comparison of two-dimensional gels showed clear differences between protein patterns of sessile and planktonic cells. Fourteen proteins increased in biofilms, whereas three decreased. From these 17 proteins, 10 were identified by MALDI-TOF-MS and could be classified into four categories according to their function: (1) general metabolism proteins (malate dehydrogenase, thiamine-phosphate pyrophosphorylase), (2) sugar and amino acid transporters (D-ribose-binding periplasmic protein, D-galactose-binding protein, YBEJ), (3) regulator proteins (DNA starvation protein and H-NS) and (4) three proteins with unknown function. The results of this study showed that E. coli O157:H7 modified the expression of several proteins involved in biofilm growth mode.     

Comparative proteome analysis of Staphylococcus aureus biofilm and planktonic cells and correlation with transcriptome profiling

Pathogenic staphylococci can form biofilms in which they show a higher resistance to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have an altered metabolic activity. Here, 2-D PAGE was used to identify secreted, cell wall-associated and cytoplasmic proteins expressed in Staphylococcus aureus after 8 and 48 h of growth. The proteins were separated at pH ranges of 4-7 or 6-11. The protein patterns revealed significant differences in 427 protein spots; from these, 258 non-redundant proteins were identified using ESI-MS/MS. Biofilm cells expressed higher levels of proteins associated with cell attachment and peptidoglycan synthesis, and in particular fibrinogen-binding proteins. Enzymes involved in pyruvate and formate metabolism were upregulated. Furthermore, biofilm cells expressed more staphylococcal accessory regulator A protein (SarA), which corroborates the positive effect of SarA on the expression of the intercellular adhesion operon ica and biofilm growth. In contrast, proteins, such as proteases and particularly immunodominant antigen A (IsaA) and staphylococcal secretory antigen (SsaA), were found in lower amounts. The RNA expression profiling largely supports the proteomic data. The results were mapped onto KEGG pathways.     

A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen Tannerella forsythia

Tannerella forsythia is a Gram‐negative anaerobe that is one of the most prominent inhabitants of the sub‐gingival plaque biofilm, which is crucial for causing periodontitis. We have used iTRAQ proteomics to identify and quantify alterations in global protein expression of T. forsythia during growth in a biofilm. This is the first proteomic study concentrating on biofilm growth in this key periodontal pathogen, and this study has identified several changes in protein expression. Moreover, we introduce a rigorous statistical method utilising peptide‐level intensities of iTRAQ reporters to determine which proteins are significantly regulated. In total, 348 proteins were identified and quantified with the expression of 44 proteins being significantly altered between biofilm and planktonic cells. We identified proteins from all cell compartments, and highlighted a marked upregulation in the relative abundances of predicted outer membrane proteins in biofilm cells. These included putative transport systems and the T. forsythia S‐layer proteins. These data and our finding that the butyrate production pathway is markedly downregulated in biofilms indicate possible alterations in host interaction capability. We also identified upregulation of putative oxidative stress response proteins, and showed that biofilm cells are 10 to 20 fold more resistant to oxidative stress. This may represent an important adaptation of this organism to prolonged persistence and immune evasion in the oral cavity.     

A simple in vitro model for growth control of bacterial biofilms

The perfused biofilm fermenter was found to be unsuitable for the long-term culture and growth rate control of Staphylococcus aureus and Pseudomonas aeruginosa biofilms. In a simplified approach, biofilms of these organisms were grown within Sorbarod filter plugs which were perfused with culture medium. Pseudo-steady states were established which were stable over several days at which the growth rate of the biofilm was reproducible, measurable and significantly slower than in broth culture. Environmental scanning electron microscopy of dissected Sorbarods demonstrated an association of cells with the surfaces of individual cellulose fibres, and growth characteristic of biofilms. Relatively high cell numbers recovered from the Sorbarod model facilitated biochemical investigations of biofilm populations and cells released spontaneously from them. SDS-PAGE demonstrated significant differences between the protein profiles of biofilm and eluted populations, which include, in Staph. aureus, the repression of a 48 kDa protein and increased expression of a 21 kDa protein relative to planktonic controls cultured at equivalent growth rates. The paper demonstrates the suitability of the approach for the culture of biofilm samples which are suitable for biochemical analysis.     

A simple in vitro model for growth control of bacterial biofilms

The perfused biofilm fermenter was found to be unsuitable for the long-term culture and growth rate control of Staphylococcus aureus and Pseudomonas aeruginosab biofilms. In a simplified approach, biofilms of these organisms were grown within Sorbarod filter plugs which were perfused with culture medium. Pseudo-steady states were established which were stable over several days at which the growth rate of the biofilm was reproducible, measurable and significantly slower than in broth culture. Environmental scanning electron microscopy of dissected Sorbarods demonstrated an association of cells with the surfaces of individual cellulose fibres, and growth characteristic of biofilms. Relatively high cell numbers recovered from the Sorbarod model facilitated biochemical investigations of biofilm populations and cells released spontaneously from them.
SDS-PAGE demonstrated significant differences between the protein profiles of biofilm and eluted populations, which include, in Staph. aureus , the repression of a 48 kDa protein and increased expression of a 21 kDa protein relative to planktonic controls cultured at equivalent growth rates. The paper demonstrates the suitability of the approach for the culture of biofilm samples which are suitable for biochemical analysis.