Streptococcus parasanguisFimbria-Associated Adhesin Fap1 Is Required for Biofilm Formation

S. parasanguis biofilm formation on polystyrene.A number of recent studies have used molecular and genetic approaches to identify genes important for biofilm formation (reviewed in reference34). One genetic approach that has been particularly fruitful in identifying genes critical for biofilm formation uses a convenient macroscopic assay to screen for biofilm-defective mutants (31). This assay is based on the ability of bacteria to form biofilms on surfaces such as polystyrene (16). Bacteria are cultured in the wells of plastic tissue culture plates, and the presence or absence of a biofilm is detected by staining the wells with crystal violet. In the present study, characterization ofS. parasanguis FW213 biofilm formation was initiated by investigating the ability FW213 to form biofilms on plastic surfaces.

Initial experiments were conducted to determine the optimal conditions for FW213 biofilm formation. Using a standard streptococcal growth medium, TH, we detected biofilms on both polystyrene (Fig.1) and polyvinyl chloride (data not shown) microtiter plates under either aerobic (Fig. 1A) or anaerobic (Fig. 1B) conditions. Although growth and biofilm formation was somewhat greater in TH under anaerobic conditions, biofilm formation was more convenient to quantify under aerobic conditions. Aerobically grown cells formed biofilms on the bottom and lower sides of the well. By contrast, anaerobically grown cells formed biofilms throughout the well, including on the side of the well above the growth medium, making it more difficult to solubilize the crystal violet-stained biofilm. Acetic acid was more effective than ethanol or ethanol-acetone (80:20) at releasing bound dye, especially from heavier biofilms (compare wells shown in Fig. 1A to those shown in Fig.2.).

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Fig. 1.

Bacterial growth and biofilm formation of S. parasanguis FW213 under different growth conditions. Cells grown in TH to early stationary growth phase were washed once in sterile water and subcultured (1:200) into either TH, TH+YE, BHI broth, CDM, TH+Glu, TSB, or TSB+YE. These cultures were then grown in polystyrene microtiter dishes at 37°C for 16 h either aerobically in 5% CO₂ (A) or anaerobically (B). Growth (light bars) and biofilm formation (dark bars) were quantified as optical density at 490 and 562 nm, respectively. Assays were performed in quadruplicate; mean values and standard deviations are shown. A representative row of crystal violet-stained microtiter plate wells is shown above each graph.

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Fig. 2.

Effect of glucose on S. parasanguis growth and biofilm formation. Cells grown in TH to early stationary growth phase were washed once in sterile water and then subcultured (1:200) into the media indicated. The percentage of glucose in each medium is given in parentheses. Cultures were grown aerobically (5% CO₂) at 37°C in polystyrene microtiter dishes for 16 h. Representative crystal violet-stained wells are shown above the graph. Growth (light bars) and biofilm formation (dark bars) are quantified below as optical density at 490 and 562 nm, respectively. Assays were performed in quadruplicate; mean values and standard deviations are shown.

Environmental factors affect S. parasanguis biofilm formation.Previous studies indicate that the nutritional content of the growth medium can regulate biofilm development (5, 26,31). In general, bacteria tend to adhere to available surfaces and form mature biofilms in environments that provide sufficient nutrients but will not adhere to surfaces in environments that are nutrient deficient. S. parasanguis FW213 biofilm formation was tested in a variety of growth media under aerobic (Fig. 1A) and anaerobic (Fig. 1B) conditions. Under both aerobic and anaerobic conditions, growth was about 1.2- and 1.6-fold greater in CDM and TH+Glu, respectively, than in TH. By contrast, biofilm formation increased significantly, about 2.6-fold in CDM and 5.1-fold in TH+Glu, under aerobic conditions but by a much smaller increment under anaerobic conditions. The final concentration of glucose in both CDM and TH+Glu is 1.0%, about four to five times greater than that in TH, BHI, or TSB.

The apparent effect of glucose on biofilm formation prompted us to compare biofilm formation of cells grown in the other types of media with and without glucose supplementation (Fig.2). In all cases, glucose enhanced biofilm formation; the most dramatic effect was with TH and BHI. This result suggests that the increased glucose promoted biofilm formation in S. parasanguis, which is in agreement with results from previous studies with other genera of bacteria (3, 31). It also supports the premise that bacteria will form biofilms under favorable nutrient conditions (5). However, there clearly are exceptions to this latter generalization. For, example, the addition of YE to TH and TSB enhanced growth of FW213 under aerobic and anaerobic conditions, but biofilm formation was not promoted by the addition of YE, a highly nutrient rich extract (Fig. 1). When added to TH, YE in fact appears to suppress biofilm formation. Biofilm formation is also suppressed in BHI, another nutrient-rich medium used to promote growth of fastidious microorganisms, including the streptococci. Other investigators have also reported that there are environmental conditions that enhance cell growth but do not promote significant biofilm formation (26,31).

Analysis of S. parasanguis mutants.A number of mutants of FW213 available in our laboratory (Table 1) were examined in the above assay to determine their ability to form a biofilm (Fig.3). In the biofilm assay, all of the mutants grew to the same extent as wild-type FW213 (Fig. 3). However, two of the mutants, VT1393 (45) and VT1428 (46), displayed severe defects in biofilm formation (Fig.3). Both mutant strains had a growth rate indistinguishable from that of wild type in liquid medium (data not shown). Both VT1393 and VT1428 contain an inactivated copy of fap1. VT1393 has an insertion mutation in the 5′ non repetitive region of fap1(45), while VT1428 has an insertion mutation at a site just upstream of the 3′ cell wall sorting signal (46). Previous studies in our laboratory have demonstrated that Fap1 is essential for formation of long fimbriae by S. parasanguisFW213 and that Fap1 is a structural subunit of the long fimbriae (45, 46). Long fimbriae are absent on the surface of Fap1-deficient strains, while monoclonal antibodies localize Fap1 to the long fimbriae and detect Fap1 as a major component of purified fimbriae (45, 46).

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Fig. 3.

Growth and biofilm formation of S. parasanguis FW213 and its isogenic mutant strains. S. parasanguis strains FW213 (wild type), VT930 (fimA), VT1393 (fap1), VT1428 (fap1), VT1346 (pepO), and VT1354 (recA) were grown in TH to early stationary growth phase, washed once in sterile water, and then subcultured (1:200) into TH+Glu. Cultures were incubated in polystyrene microtiter dishes at 37°C, 5% CO₂ for 17 h. Crystal violet-stained wells are shown above the graph. Growth (light bars) and biofilm formation (dark bars) are quantified below as optical density at 490 and 562 nm, respectively. Assays were performed in quadruplicate; mean values and standard deviations are shown.

Our current findings, using two independent mutant alleles offap1, indicate that Fap1 plays an important role in S. parasanguis FW213 biofilm formation. Thus, the long peritrichous fimbriae, comprised of Fap1, may not be limited to the mediation of cell-to-surface interactions on the tooth surface but may be involved in the mediation of cell-to-surface interactions on solid surfaces in general. It has been demonstrated in other bacterial species that fimbriae (or pili) are required for biofilm formation (30, 33,43, 44).

A mutant defective in the production of FimA, another protein associated with FW213 fimbriae, was capable of biofilm formation (Fig.3). The fimA gene encodes the lipoprotein portion of an ATP binding cassette transporter (15) that belongs to a family of transporters involved with the uptake of metal cations (8). While FimA is a virulence factor of native valve endocarditis and facilitates binding to fibrin (1), it does not appear to play a role in biofilm formation under the environmental conditions used in this study. FimA is not required for the assembly of FW213 fimbriae or for adhesion to SHA (14).

An isogenic strain of FW213 defective in RecA function (20) was capable of biofilm formation (Fig. 3) as might be expected. Likewise, a strain defective in PepO endopeptidase activity (19) formed a biofilm in the above assay. S. parasanguis PepO is similar in structure and activity to mammalian enzymes that play essential roles in events such as inflammatory response phenomena, pain, and cardiovascular regulation.

Time course assay of biofilm development of wild-type FW213 and of a fap1 mutant.The difference in biofilm formation between wild-type FW213 and the fap1 mutants prompted us to study biofilm development in these strains in more detail. Biofilm development by both wild-type FW213 and the fap1 mutant VT1393 was studied over a period of 14 h (Fig.4). The wild-type strain displayed a time-dependent increase in biofilm formation that reached a plateau, as detected by crystal violet staining after about 10 h. By contrast, FW213 cells lacking Fap1, and hence the long peritrichous fimbriae, were severely hindered in the ability to form biofilms and displayed only low levels of crystal violet staining over the course of the experiment. The apparent differences in biofilm-forming competency could not be explained by unequal growth, as growth of thefap1 mutant was equivalent to that of the wild type at each time point (data not shown).

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Fig. 4.

Biofilm development over 14 h for S. parasanguis FW213 and fap1 mutant VT1393. Cells were grown in TH to early stationary growth phase, washed once in sterile water, and diluted 1:50 in TH+Glu. Cells were then grown in polystyrene microtiter dishes, and at the times indicated, biofilm formation was assayed. (A) Representative crystal violet-stained wells at each time point for FW213 and the fap1 mutant. (B) Quantification of crystal violet staining. Assays were performed in quadruplicate; mean values and standard deviations are shown.

Microscopic analysis of wild-type and fap1 mutant biofilms.The detection of biofilm formation in the above studies was done indirectly by determining the amount of crystal violet-stained cells that attach to wells of polystyrene microtiter dishes. Biofilm formation, however, is considered a complex developmental process (5, 7, 30, 31, 44) that can be broken down into a series of observable steps (5, 44). These steps are likely to include attachment and immobilization on a surface, cell-to-cell attachment, microcolony formation, formation of a confluent biofilm, and formation of a characteristic three-dimensional biofilm structure. To outline steps involved in biofilm formation by S. parasanguis FW213, we used an experimental system for the development of three-dimensional biofilms as reported by Watnick and Kolter (44). We also used this system to help identify the role(s) of Fap1 and fimbriae during FW213 biofilm formation.

Biofilm development on vinyl coverslips by wild-type FW213 or thefap1 mutant VT1393 was observed microscopically at 1, 2, 3, 6, 15, and 18 h after inoculation. Images taken at 1, 3, and 18 h after inoculation are presented in Fig.5. Each image is a representative sample of what was observed in multiple fields. After 1 h of incubation (Fig. 5A), numerous cells of the wild-type strain had attached to the plastic coverslip and several small clusters had formed. Fewer of thefap1 mutant cells had attached to the coverslip after 1 h of incubation, and no small cell clusters were seen (Fig. 5D). After 3 and 18 h of incubation, the patterns of attachment seen for wild-type FW213 (Fig. 5B and C) and the fap1 mutant (Fig. 5E and F) were quite different. Many more wild-type S. parasanguis FW213 cells had attached to the coverslip, and larger clusters of cells had formed. By 18 h, the wild-type biofilm consisted of multiple layers of cells and displayed dark clusters of cells (microcolonies) interspersed with areas of less densely packed cells. In contrast, the fap1 mutant strain displayed broad empty areas of plastic surface with only a few cells scattered throughout. No cell clusters or microcolonies were evident. These data are consistent with the indirect analyses of biofilm formation using crystal violet staining shown in Fig. 3 and 4. They demonstrate that Fap1 and fimbriae play a critical role in FW213 biofilm formation.

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Fig. 5.

Phase-contrast micrographs comparing biofilm formation of wild-type S. parasanguis FW213 and the fap1mutant VT1393 on plastic coverslips over time. (A to C) FW213 biofilms taken at 1, 3, and 18 h after inoculation, respectively; (D to F)fap1 mutant biofilms taken at comparable times. The arrow indicates one of many microcolonies that form over the course of the experiment. Bar = 30 μm.

Effect of glucose on surface expression of Fap1.Given our findings that Fap1 and fimbriae play a role in FW213 biofilm formation and that glucose promotes biofilm formation in FW213, we asked whether glucose concentration in the growth medium affected the surface expression of Fap1 and fimbriae. Utilizing a bactELISA with a monoclonal antibody specific to Fap1, we determined that an increase in glucose concentration did not increase surface expression of Fap1. This result suggests that factors other than Fap1 and fimbriae were responding to the increased glucose concentration and were contributing to FW213 biofilm formation in these experiments.

Although environmental signals such as carbon source availability play a role in regulating biofilm formation, little is known about the molecular mechanisms that link environmental signals and biofilm development. However, a recent study has demonstrated that the global carbon metabolism regulator Crc of Pseudomonas aeruginosa is a component of a signal transduction pathway required for biofilm formation (29). Crc senses carbon source availability and subsequently affects expression of the type IV pilA gene. Since type IV pilus-mediated twitching motility is required forP. aeruginosa biofilm formation, Crc appears to tie nutritional cues to the regulation of biofilm development.

S. parasanguis FW213 belongs to a group of gram-positive bacteria with low GC content. In this group of bacteria, carbon catabolite repression is mediated by CcpA (38). It would be interesting to determine whether S. parasanguis FW213 possesses CcpA and, if so, whether CcpA serves as a regulator that links carbon availability to biofilm formation in this organism.

While glucose may serve as a nutritional signal for biofilm formation in S. parasanguis FW213, an increase in glucose concentration may possibly signal a change in the osmolarity of the environment. Previous studies suggest that bacteria encounter conditions of higher osmolarity both as they approach a solid surface and as they exist within a biofilm. These changes in osmolarity result in the expression of surface- and biofilm-specific genes (31,35). FW213 may perceive the increase in glucose concentration as an increase in osmolarity, thus triggering the expression of additional genes required for biofilm formation. An increase in sucrose concentration also promoted FW213 biofilm formation (data not shown), giving additional support to the hypothesis that osmolarity may be affecting biofilm formation. At some point, however, increases in osmolarity begin to inhibit biofilm formation (26, 31).

An increase of glucose or sucrose concentration in TH broth affects the final pH of the medium after bacterial growth (data not shown). Thus, localized changes in pH maybe also be important in biofilm formation. However, in other organisms, no effect on biofilm formation is seen over a range of pH. With Pseudomonas fluorescens, no effect on biofilm formation was seen in media ranging from pH 5 to 8.5 (31). Similarly, Streptococcus gordonii biofilm formation was unaffected in media ranging from pH 6 to 10.5 (26). Additional studies are required to elucidate the relationship of carbon source availability, osmolarity, and pH to biofilm formation in S. parasanguis FW213.

Conclusions.This study was undertaken to investigate whether S. parasanguis FW213 can form biofilms in vitro and to determine some of the environmental parameters that affect biofilm formation. It was determined thatS. parasanguis FW213 can form biofilms on solid surfaces, such as polystyrene and polyvinyl-chloride, under a range of growth conditions. Glucose promoted biofilm formation in FW213, suggesting that glucose is an environmental signal regulating FW213 biofilm development. This result supports the idea that bacteria form biofilms under favorable nutrient conditions. It does not rule out, however, the possibility that other factors associated with an increase in glucose concentration, such as pH and/or osmolarity, play a role in biofilm formation.

We demonstrated that those FW213 strains deficient in the production of Fap1, and hence in the production of fimbriae, are defective in the ability to form biofilms on a solid surface. Discrete stages of biofilm formation of both the wild type and the fap1 mutant on polystyrene were examined via phase-contrast microscopy. Differences in initial attachment and in the progression of biofilm formation over time indicate that Fap1 and fimbriae play a role in early cell-to-surface interactions during biofilm formation. The fewfap1 mutant cells attached to the surface never formed cell aggregates or microcolonies. Further studies are needed to determine whether Fap1 and fimbriae also play a role in cell-to-cell interactions.

Currently the biofilm assay is being used in studies aimed at identification of additional genes necessary for biofilm initiation and/or development in S. parasanguis FW213. Understanding critical steps involved in biofilm formation and metabolism may suggest new therapies for treatment or prevention of biofilm-related infections.