Role of cell surface hydrophobicity in Candida albicans biofilm

Overall cell surface hydrophobicity (CSH) is predicted to play an important role during biofilm formation in Candida albicans but is the result of many expressed proteins. This study compares the CSH status and CSH1 gene expression in C. albicans planktonic cells, sessile biofilm, and dispersal cells. Greater percentages of hydrophobic cells were found in non-adhered (1.5 h) and dispersal forms (24 or 48 h) (41.34±4.17% and 39.52±7.45%, respectively), compared with overnight planktonic cultures (21.69±3.60%). Results from quantitative real-time PCR confirmed greater up-regulation of the CSH1 gene in sessile biofilm compared with both planktonic culture and dispersal cells. Up-regulation was also greater in dispersal cells compared with planktonic culture. The markedly increased CSH found both in C. albicans biofilm, and in cells released during biofilm formation could provide an advantage to dispersing cells building new biofilm.     

Effect of biofilm growth on expression of surface proteins of Actinomyces naeslundii genospecies 2

The predominant surface proteins of biofilm and planktonic Actinomyces naeslundii, a primary colonizer of the tooth surface, were examined. Seventy-nine proteins (the products of 52 genes) were identified in biofilm cells, and 30 of these, including adhesins, chaperones, and stress-response proteins, were significantly up-regulated relative to planktonic cells.     

Protein Expression by Streptococcus mutans during Initial Stage of Biofilm Formation

Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface. Log-phase planktonic cells of S. mutans were allowed to adhere to a glass slide for 2 h in the presence of a ¹⁴C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished ≥1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.     

The use of glass wool as an attachment surface for studying phenotypic changes in Pseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis was used to demonstrate phenotypic differences between Pseudomonas aeruginosa biofilm cells and the planktonic counterpart cells under defined culture conditions. Glass wool was used as a substratum for cell attachment as it affords a large surface-to-volume ratio (1 g with a mean diameter of 15 microns = 1300 cm2), supports the growth of biofilms, allows for free movement of cells between the inter-strand spaces, and it facilitates the exchange of nutrients and oxygen. It also allows for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterization. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells. We selected 41 proteins, the levels of which varied in a significant and reproducible way in the respective protein profiles. In the biofilm cells, a general up-regulation of the spots was seen, but in SIP cells expression of these spots were generally down-regulated. Altogether six unique proteins were seen in the planktonic cells, while the biofilm and SIP cells contained five and two unique proteins, respectively. Glass wool, therefore, appears to be an ideal attachment surface for the study of biofilm development.     

Chemical composition of Enterococcus faecalis in biofilm cells initiated from different physiologic states

Enterococcus faecalis is a ubiquitous bacterium of the gut that is observed in persistent periradicular infections. Its pathogenicity is associated with biofilm formation and the ability to survive under nutrient-poor (starvation) conditions. However, characteristics of chemical composition of biofilm cells developed by starved E. faecalis cells remain poorly understood. In this study, E. faecalis cells in exponential, stationary, and starvation phases were prepared and separately cultured to form biofilms. Confocal laser scanning microscopy was performed to verify biofilm formation. Raman microscopy was used to investigate the chemical composition of cells within the biofilms. Compared to cells in exponential or stationary phase, starved cells developed biofilms with fewer culturable cells (P?E. faecalis.     

Surface proteins and the formation of biofilms by Staphylococcus aureus

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12–24 h after stationary phase, and more mature biofilms formed for up to 60 h after stationary phase. All samples were labeled either by (i) [¹⁵N]glycine and l-[1-¹³C]threonine, or in separate experiments, by (ii) l-[2-¹³C,¹⁵N]leucine. We then measured ¹³C-¹⁵N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in ¹⁵N isotopic enrichments and from the routing of ¹³C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion.     

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.     

Biofilm-induced modifications in the proteome of Pseudomonas aeruginosa planktonic cells

While recent studies focused on Quorum Sensing (QS) role in the cell-to-cell communication in free or biofilm cultures, no work has been devoted up to now to investigate the communication between sessile and planktonic bacteria. In this aim, we elaborated an original two-chambered bioreactor and used a proteomic approach to study the alterations induced by Pseudomonas aeruginosa biofilm cells on protein expression in planktonic counterparts (named SIPs for Surface-Influenced Planktonics). Proteomic analyses revealed the existence of 31 proteins whose amount varied in SIPs, among which five corresponded to hypothetic proteins and two (the Fur and BCP proteins) are involved in bacterial response to oxidative stress. An increase in the concentration of C₄-HSL (rhlR–rhlI-dependent QS) and 3-oxo-C₁₂-HSL (lasR–lasI-dependent QS) autoinducer molecules was shown in the planktonic compartment. Interestingly, among proteins that were accumulated by SIPs was 3-oxoacyl-[acyl-carrier-protein] reductase, a protein involved in the production of the autoinducer 3-oxo-C₁₂-HSL. These results demonstrate that planktonic organisms are able to detect the presence of a biofilm in their close environment and to modify their gene expression in consequence.     

Non-glucan Attached Proteins of Candida albicans Biofilm Formed on Various Surfaces

Non-glucan attached proteins of the cell surface and extracellular matrix of Candida albicans biofilms formed on two catheter surfaces and denture acrylic were examined. The SDS-PAGE protein profiles of these proteins compared with that obtained from planktonic yeast cells and germ tubes were generally similar. This observation suggested that this class of biofilm surface proteins is not composed of a unique set of extracellular proteins or that one or a few proteins dominate the non-glucan attached proteins of biofilm. However, differences were observed in the proteins obtained from biofilm formed on one catheter surface and two proteins, Grp2p and ORF19.822p, identified by mass spectrometry following two-dimensional separation. These proteins have previously been associated with drug resistance and their presence or abundance appeared to be influenced by the surface on which the biofilm was formed.     

Antigen expression in biofilm cells of Aeromonas hydrophila employed in oral vaccination of fish

Total protein, S-layer protein and lipopolysaccharides (LPS) of biofilm cells of Aeromonas hydrophila were analysed by SDS-PAGE and compared with that of planktonic cells. In the whole cell lysate of biofilm cells, about 15 proteins were repressed while three new proteins were expressed compared to that in planktonic cells. Interestingly, in biofilm cells the S-layer proteins were lost and LPS showed an additional high molecular weight band compared to that in planktonic cells. We propose that the change in LPS profile must have contributed to the loss of S-layer. Also, the high molecular weight band of LPS might play a role in the better performance of biofilm oral vaccine by eliciting a protective immune response.     

Irradiation Sensitivity of Planktonic and Biofilm-Associated Escherichia coli O157:H7 Isolates Is Influenced by Culture Conditions

Ionizing radiation effectively inactivates Escherichia coli O157:H7, but the efficacy of the process against biofilm cells versus that against free-living planktonic cells is not well documented. The radiation sensitivity of planktonic or biofilm cells was determined for three isolates of E. coli O157:H7 (C9490, ATCC 35150, and ATCC 43894). Biofilms were formed on sterile glass slides incubated at 37°C for either 24 h, 48 h, or 72 h. The biofilm and planktonic cultures were gamma irradiated at doses ranging from 0.0 (control) to 1.5 kGy. The dose of radiation value required to reduce the population by 90% (D₁₀) was calculated for each isolate, culture, and maturity based on viable populations at each radiation dose. For each of the times sampled, the D₁₀ values of isolate 43894 planktonic cells (0.454 to 0.479 kGy) were significantly (P < 0.05) higher than those observed for biofilm cells (0.381 to 0.385 kGy), indicating a significantly increased sensitivity to irradiation for cells in the biofilm habitat. At the 24-h sampling time, isolate C9490 showed a similar pattern, in which the D₁₀ values of planktonic cells (0.653 kGy) were significantly higher than those for biofilm cells (0.479 kGy), while isolate 35150 showed the reverse, with D₁₀ values of planktonic cells (0.396 kGy) significantly lower than those for biofilm cells (0.526 kGy). At the 48-h and 72-h sampling times, there were no differences in radiation sensitivities based on biofilm habitat for C9490 or 35150. Biofilm-associated cells, therefore, show a response to irradiation which can differ from that of planktonic counterparts, depending on the isolate and the culture maturity. Culture maturity had a more significant influence on the irradiation efficacy of planktonic cells but not on biofilm-associated cells of E. coli O157:H7.     

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.     

Physiochemical and molecular properties of antimicrobial-exposed Staphylococcus aureus during the planktonic-to-biofilm transition

This study was designed to characterize the physicochemical and molecular properties of Staphylococcus aureus cells treated with nisin, allyl isothiocyanate (AITC), thymol, eugenol, and polyphenol during the transition from planktonic to biofilm growth as measured by hydrophobicity, auto-aggregation, and differential gene expression. Thymol exhibited the highest antimicrobial activity against planktonic, biofilm-forming, biofilm, and dispersed cells, showing 0.21, 0.22, 0.46, and 0.26 mg/ml of MIC values, respectively. The lowest hydrophobicity was observed in planktonic cells treated with polyphenol (16 %), followed by thymol (29 %). The auto-aggregation abilities were more than 85 % for nisin, AITC, eugenol, polyphenol, and the control. The cell-to-surface interaction was related positively to biofilm formation by S. aureus. The adhesion-related gene (clfA), virulence-related genes (spa and hla), and efflux-related gene (mdeA) were down-regulated in both planktonic and biofilm cells treated with AITC, thymol, and eugenol. The results suggest that the antimicrobial tolerance and virulence potential were varied in the cell states during the planktonic-to-biofilm transition. This study provides useful information for understanding the cellular and molecular responses of planktonic and biofilm cells to antimicrobial-induced stress.     

Protein expression by planktonic and biofilm cells of Streptococcus mutans

Streptococcus mutans, a major causal agent of dental caries, functions in nature as a component of a biofilm on teeth (dental plaque) and yet very little information is available on the physiology of the organism in such surface-associated communities. As a consequence, we undertook to examine the synthesis of proteins by planktonic and biofilm cells growing in a biofilm chemostat at pH 7.5 at a dilution rate of 0.1 h(-1) (mean generation time=7 h). Cells were incubated with (14)C-labelled amino acids, the proteins extracted and separated by two-dimensional electrophoresis followed by autoradiography and computer-assisted image analysis. Of 694 proteins analysed, 57 proteins were enhanced 1.3-fold or greater in biofilm cells compared to planktonic cells with 13 only expressed in sessile cells. Diminished protein expression was observed with 78 proteins, nine of which were not expressed in biofilm cells. The identification of enhanced and diminished proteins by mass spectrometry and computer-assisted protein sequence analysis revealed that, in general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while biosynthetic processes were enhanced. The results show that biofilm cells possess novel proteins, of as yet unknown function, that are not present in planktonic cells.     

Protein expression by Streptococcus mutans during initial stage of biofilm formation

Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface. Log-phase planktonic cells of S. mutans were allowed to adhere to a glass slide for 2 h in the presence of a (14)C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished > or =1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.     

Application of 16O/18O reverse proteolytic labeling to determine the effect of biofilm culture on the cell envelope proteome of Porphyromonas gingivalis W50

Porphyromonas gingivalis is an oral pathogen linked to chronic periodontitis. The bacterium exists as part of a polymicrobial biofilm accreted onto the tooth surface. An understanding of the changes to the proteome especially of the cell envelope of biofilm cells compared with planktonic cells could provide valuable insight into the molecular processes of biofilm formation. To establish which proteins changed in abundance between the planktonic and biofilm growth states, the cell envelope fractions of two biological replicates of P. gingivalis cultivated in a chemostat were analysed. Proteins were separated by 1-D SDS-PAGE, in-gel digested with trypsin in the presence of H216O or H218O and identified and quantified by LC-MALDI TOF/TOF-MS. Using a reverse labeling strategy we identified and quantified the changes in abundance of 81 P. gingivalis cell envelope proteins. No form of bias between the labels was observed. Twenty four proteins increased in abundance and 18 decreased in abundance in the biofilm state. A group of cell-surface located C-Terminal Domain family proteins including RgpA, HagA, CPG70 and PG99 increased in abundance in the biofilm cells. Other proteins that exhibited significant changes in abundance included transport related proteins (HmuY and IhtB), metabolic enzymes (FrdAB) and immunogenic proteins.     

Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa

A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa. A rotating-disk biofilm reactor was used to generate biofilm and free-swimming cultures to test their relative levels of resistance to heavy metals. It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells. However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells. Microscopy was used to evaluate the effect of copper stress on a mature P. aeruginosa biofilm. The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum. A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.