Heparin Mimics Extracellular DNA in Binding to Cell Surface-Localized Proteins and Promoting Staphylococcus aureus Biofilm Formation

Heparin enhances biofilm formation in multiple strains of S. aureus and coagulase-negative staphylococci.Shanks and coworkers reported that the addition of 1,000 U/ml of sodium heparin to the growth medium stimulated biofilm formation in S. aureus strain MZ100 and a few other related strains (9). To determine the prevalence of this phenomenon, the effect of heparin on biofilm formation was tested in various clinical isolates of S. aureus, including both methicillin-susceptible S. aureus (MSSA) and MRSA strains. A total of 9 strains of S. aureus were analyzed for biofilm formation in the presence of 100 U/ml of sodium heparin or ammonium heparin in an in vitro biofilm assay (17). A dose of 100 U/ml is a clinically relevant dose of sodium heparin and was used in all further experiments in this work unless mentioned otherwise (18). Ammonium heparin chemically resembles sodium heparin, and it was included in most of the experiments to rule out the effect of sodium addition alone. In biofilm microtiter assays, heparin stimulated increase in biofilm biomass for seven out of nine strains (Fig. 1A), and the phenomenon was spread across different strain lineages. Strains showing heparin-mediated increase in the assay also showed pronounced aggregation of cells when grown overnight in culture tubes (see Fig. S1 in the supplemental material). A few strains did not exhibit enhanced biofilm formation in the presence of heparin (Fig. 1A, strains MW2 and UAMS1), and these strains aggregated poorly (Fig. S1).

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FIG 1

Heparin enhances S. aureus and coagulase-negative staphylococcus (CoNS) biofilm formation. (A and B) Microtiter biofilms of various S. aureus strains (A) or various CoNS species (B), grown in the presence or absence of sodium heparin (sod hep) or ammonium heparin (amm hep) as indicated. Values are the means ± standard deviations (error bars) from three independent experiments, with four technical replicates in each experiment. (C) Dose dependence of biofilm formation by strain LAC in the presence of increasing amounts of sodium heparin. A Student t test was performed, and statistical significance is indicated as follows: *, P < 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005.

Analysis of the effects of heparin on biofilm formation was further extended to coagulase-negative staphylococci (CoNS), including Staphylococcus epidermidis, S. lugdunensis, S. hominis, S. haemolyticus, S. saprophyticus, and S. warneri. There is increasing incidences of device-related infections of S. epidermidis and other related CoNS species (19), and thus, we extended our analysis of heparin impact to these CoNS. S. epidermidis, S. haemolyticus, and S. hominis showed a 90% to 400% increase in biofilm biomass with heparin supplementation, whereas S. lugdunensis, S. warneri, and S. saprophyticus remained unaffected under the conditions used (Fig. 1B). Taken together, these results suggest that heparin-mediated increase in biofilm biomass is a widespread phenomenon among various strains of S. aureus and CoNS.

To further investigate the heparin enhancement mechanism, a community-associated MRSA (CA-MRSA) isolate of the USA300 lineage (strain LAC) was used in this work. This strain was chosen because of its clinical significance and the availability of existing tools and a transposon mutant library (20). Reports of isolation of USA300 in biofilm infections such as infective endocarditis, osteomyelitis, and prosthetic joint infections have increased (21–23), suggesting these strains are relevant for study of biofilm mechanisms. As noted above, LAC biofilm capacity is enhanced with sodium or ammonium heparin (Fig. 1A), and further testing showed a dose-dependent increase in biofilm biomass with sodium heparin that plateaued at about 100 U/ml (Fig. 1C).

Heparin does not impact S. aureus growth or pH.A series of initial experiments were carried out to investigate the mechanism of heparin enhancement of biofilms. One possible mechanism is through heparin improvement of S. aureus growth, since bacteria have been observed to degrade and grow on heparin (24). However, heparin supplementation did not affect growth of the wild-type LAC strain (Fig. S2), suggesting that heparin impacts some phase of biofilm development. For in vitro studies, glucose is a known additive that positively impacts S. aureus biofilm formation (25), and this occurs through a decrease in the pH of the medium due to acetate excretion (26), repressing the agr quorum-sensing regulon (26). However, heparin supplementation in tryptic soy broth (TSB) did not lower the pH, even after biofilms were allowed to form for 18 h (Table 1). The only major changes in pH observed in the biofilm medium with various S. aureus strains were due to glucose supplementation.

Heparin increases biofilm cell retention but does not initiate attachment.Biofilms consist of aggregates of bacteria attached to a surface and held together in a complex extracellular matrix (5, 27). We hypothesized that heparin may alter the properties of the matrix, leading to enhanced retention of cells that results in a thicker, more dense biofilm structure. To test this hypothesis, the number of CFU within the biofilm was determined and compared to the number of planktonic cells surrounding the biofilm, with and without heparin supplementation. Importantly, the number of CFU increased up to 50% in the presence of sodium heparin, while the total number of cells (both planktonic and biofilm) remained unaffected (Fig. 2A). This increase in biofilm biomass and CFU was independent of glucose supplementation (data not shown). These data suggest that heparin retains S. aureus cells within the biofilm matrix at the expense of neighboring planktonic cells.

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FIG 2

Heparin enhances cell retention within a biofilm but does not promote attachment. (A) The fraction of planktonic cells compared to biofilm cells was determined by suspending either the entire contents of a microtiter well (total) or just the biofilm after removal of spent medium (biofilm). When heparin was present, the number of planktonic cells was negligible compared to the number of cells in the biofilm. (B) The wells of a polystyrene or glass-bottom microtiter plate were incubated with 100 U/ml of heparin in PBS for 24 h [indicated by “(c)” (for coated)], washed to remove free heparin, and inoculated with strain LAC. After 18 h of incubation, biofilm biomass was assessed. As a negative control, wells were preincubated with PBS, and as a positive control, heparin was added at the time of inoculation with the LAC strain. (C) To assess the kinetics of heparin enhancement of biofilm formation, heparin was added at the time of inoculation (0 h) or after 18 h of growth in the absence of heparin (18 h). Biofilm biomass was determined after one additional hour of incubation. (D) Effect of washing on heparin-dependent biofilm formation. Strain LAC was grown for 18 h in the absence of heparin, at which time the cells were washed to remove secreted proteins and then transferred to the wells of a microtiter plate. Heparin was added, and biofilm biomass was assessed after 1 h. For comparison, the assay was also performed using the planktonic cells directly (unwashed). A glucose-only control (glc) received no heparin. Values in each panel are the averages ± standard deviations from three independent experiments. Statistical significance (*, P < 0.05; **, P < 0.005) was determined by a Student t test and is based on comparison to the value for the untreated control for each condition.

Biofilm formation begins with bacterial attachment to a surface, and the positive impact of heparin on S. aureus biofilms could be due in part to improved cell attachment. This question was tested by coating microtiter wells with sodium heparin (100 U/ml), washing with phosphate-buffered saline (PBS) to remove free heparin, and then inoculating with strain LAC to initiate biofilm development. Control wells were set up in parallel with an identical concentration of heparin added to the medium at the time of inoculation. As shown in Fig. 2B, the heparin-dependent increase in biofilm biomass was observed only in the control wells, suggesting that heparin does not facilitate direct bacterial attachment to the microtiter plates. Similar results were obtained using glass plates (Fig. 2B), indicating that altering the surface chemistry does not change the observation. Taken together, heparin improves S. aureus cell retention within a biofilm but does not enhance surface attachment.

To further investigate these observations, biofilms were allowed to establish before heparin supplementation. Remarkably, heparin still increased biofilm biomass within 1 h of addition (Fig. 2C). This observation held true even if chloramphenicol was added to inhibit protein synthesis just prior to heparin addition (data not shown), suggesting that heparin does not induce production of biofilm-related proteins. Further support for this interpretation came from experiments in which S. aureus was grown for 18 h in culture tubes and transferred to the wells of a microtiter plate. The addition of heparin to these cell suspensions promoted biofilm formation within 1 h (Fig. 2D). If the cells were washed before transfer to the microtiter plate, heparin did not enhance biofilm formation (Fig. 2D). Washing not only affects the pH of the medium but also removes proteins from the extracellular environment. Collectively, these findings are consistent with a model in which heparin facilitates the rapid capture of planktonic cells from the surrounding liquid phase and incorporates them into the biofilm. The negative impact of washing suggests extracellular proteins might be involved, and this question is further addressed below.

Heparin localizes within the biofilm matrix.The model described above predicts that heparin is incorporated into the biofilm extracellular matrix. To test this idea, a green fluorescent protein (GFP)-expressing LAC strain was allowed to grow to stationary phase (18 h), at which time rhodamine-labeled heparin (rhodamine-heparin) was added. After an additional hour of incubation, biofilms were examined by confocal microscopy. As predicted, rhodamine-heparin was found to accumulate around the green fluorescent LAC strain (Fig. 3A). Biofilms that lacked exogenously added rhodamine-heparin showed only green fluorescence (Fig. 3B). The importance of heparin as a potential matrix component was further tested using heparinase (heparin lyase), which catalyzes breakdown of heparin into constituent disaccharides. Initially, heparinase was added together with heparin at the time of inoculation. In this case, heparinase limited the enhancement of biofilm formation by heparin (Fig. 3C), presumably due to breaking down before a positive impact on the biofilm could be observed. If heparin was added and the biofilm was allowed to establish, the addition of heparinase had no effect (Fig. 3C). In a mature biofilm, it is possible that heparin is inaccessible to the enzyme, or potentially an established biofilms is not affected by the removal of heparin from the matrix. With evidence that heparin is incorporated into the matrix, these observations raise questions of whether it substitutes for, or works together with, other components of the matrix.

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FIG 3

Heparin localizes in the S. aureus biofilm matrix. Confocal laser scanning microscopic images of LAC sGFP biofilm with (A) or without (B) rhodamine-heparin. (C) Effect of heparinase on biofilm biomass. Strain LAC was allowed to form biofilms for 18 h in the absence (−) (control) or presence of heparin. Where indicated, heparinase was added at the time of inoculation (0 h) or after allowing biofilms to form for 17 h. Values are the averages ± standard deviations from three independent experiments. Statistical significance (*, P < 0.05) was determined by a Student t test.

Heparin substitutes for eDNA in the biofilm matrix. S. aureus biofilms have an extracellular matrix that consists of polysaccharides, proteins, and eDNA (5, 27–30). Evidence in favor of eDNA as an important matrix component came in part from studies on the role of secreted nuclease in biofilm development. A mutant defective in the nuc gene, which encodes the major secreted nuclease in strain LAC, forms larger biofilms and accumulates more eDNA in the matrix than the wild type does (31, 32). Heparin is a large polyanionic polymer like eDNA and potentially could mimic the function of eDNA in the biofilm matrix. The observed heparin interaction with surface-localized proteins (Fig. 2D and 3), and positive impact on bacterial aggregation (Fig. S1), are consistent with this line of reasoning. Therefore, we hypothesized that heparin-dependent biofilm would be affected by modulating the level of eDNA in the matrix. To test this hypothesis, the effect of heparin on biofilm formation was assayed in nuclease mutants, which accumulate more eDNA than wild-type strains do (31). A LAC nuc mutant exhibited more-robust biofilms than the wild type did, consistent with previous reports (31), but the addition of heparin did still lead to a further increase in biofilm biomass (Fig. 4). Conversely, a LAC nuc2 mutant, which lacks the additional surface-localized nuclease (33), formed a biofilm like the wild-type-like biofilm that was significantly enhanced by exogenous heparin. This observation is consistent with previous reports that mutations in nuc2 are not linked to biofilm formation (33). When both nuc and nuc2 were removed, maximizing eDNA levels in the matrix, heparin biofilm enhancement was reduced (Fig. 4) and no longer reached significance with ammonium heparin. These findings support a model in which heparin and eDNA can substitute for one another in the biofilm matrix, both leading to a positive impact on biofilm biomass.

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FIG 4

Effect of eDNA on heparin-mediated biofilm formation. Effect of heparin on biofilm formation in mutants defective in one or both nucleases (nuc and nuc2) was compared with the wild-type LAC strain. Three independent biological experiments were performed, each with four technical replicates, and the error bars represent the standard deviation of the entire data set. Statistical significance (*, P < 0.05) was determined by a Student t test.

Proteins are required for heparin-dependent biofilm formation.On the basis of the results of the washing experiments (Fig. 2D), we predicted that surface and/or extracellular proteins are involved in heparin biofilm enhancement. To address this question, the sensitivity of heparin biofilms to proteinase K treatment was tested. Notably, proteinase K treatment completely disintegrated the biofilm containing heparin, regardless of whether heparin was added at the time of inoculation or after biofilms had been allowed to form for 18 h (Fig. 5A). Proteinase K also disintegrated the biofilm in the control wells that lacked heparin, suggesting that the biofilm of the LAC strain is mostly held together by proteins, as shown previously (34, 35). This finding was further investigated using a LAC mutant lacking all secreted proteases (AH1919 strain, Δproteases) (36, 37), which formed a more robust biofilm than the wild-type strain did (Fig. 5B), presumably by eliminating self-cleavage of surface/extracellular proteins. The biofilm of the protease deficient strain was further increased by heparin addition. This observation suggests that heparin does not stimulate biofilm biomass by inhibiting secreted protease activity. To gain further information on the proteins involved in binding heparin, wild-type LAC (JE2 version) was compared to a knock out in sortase A (Fig. 5C). The srtA::Tn mutant still formed a biofilm with glucose, but the enhancement with heparin (either sodium or ammonium forms) was reduced compared to strain JE2, which suggests that sortase-anchored proteins may have some contribution to the heparin phenotype. Taken together, the heparin biofilm enhancement depends on S. aureus surface/extracellular proteins, and the phenotype is independent of known secreted proteases.

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FIG 5

Proteins mediate heparin-dependent biofilm formation. (A) Proteinase K treatment abolishes biofilms whether added at the time of inoculation (0 h) or after allowing biofilms to form for 17 h. (B) Strain AH1919, a LAC derivative lacking 10 secreted proteases (Δprotease), forms a more robust biofilm than the wild-type parent LAC did. The biofilms for both strains were also compared in the presence of sodium heparin and ammonium heparin as indicated. (C) Wild-type (WT) strain JE2 biofilms were compared to srtA::Tn mutant in the presence or absence of sodium and ammonium heparin as indicated. For each panel, the values are the averages ± standard deviations from three independent experiments, each with four technical replicates. Statistical significance (*, P < 0.05; **, P < 0.005) was determined by a Student t test and is based on comparison to the value for the untreated control for each condition.

Identification of heparin-binding proteins by mass spectrometry.In order to identify the matrix proteins that interact with heparin, the wild-type LAC strain was grown in biofilm media containing 100 U/ml of sodium heparin for 18 h. Heparin-binding proteins were identified separately in the culture supernatant and cell wall fractions. Briefly, the culture was centrifuged to pellet the cells, and additional heparin was added to the supernatant fraction to capture secreted heparin-binding proteins. Protoplasts were prepared, and heparin was added to the supernatant to capture released cell wall proteins. Avidin-agarose beads were used to precipitate heparin from the supernatant and cell wall fractions, and associated proteins were eluted, separated by SDS-PAGE, and visualized by silver staining. Isolated bands were excised from the gel, digested with trypsin in situ, and analyzed by mass spectrometry. Using 15% sequence coverage as a cutoff, peptides from more than 40 secreted proteins and 100 cell wall proteins were identified (Table 2). Peptides corresponding to nine of these proteins appeared in both fractions. The majority of the proteins identified in the cell wall fraction were cytoplasmic; however, some of the cytoplasmic proteins have been reported to localize on the cell wall in the biofilms (38–40).

The most abundant protein in the secreted fraction was bifunctional autolysin (Atl), which is the major peptidoglycan hydrolase in S. aureus (41). Atl was followed by protein A in the secreted fraction, and protein A was the most abundant protein in the cell wall fraction. Protein A is a cell wall-anchored (CWA) protein that is shed at high levels (42, 43), and it contains four or five homologous modules that can bind to multiple ligands (44). Apart from protein A, four other cell wall-anchored proteins were detected in the secreted fraction: penicillin-binding protein 2′ (PBP2′), SasG (SAUSA300_2436), a putative surface protein (SAUSA300_0883), and another surface protein (SAUSA300_2164). SasG is a known CWA surface protein that promotes biofilm formation by S. aureus (45). Interestingly, SasG (SAUSA300_2436) is truncated after 444 amino acids in the LAC strain, while SasG in other strains is intact at ~1,630 amino acids, depending on the strain. PBP2′ is present only in the MRSA strains and has been observed to affect biofilm formation in MRSA strain BH1CC (46). SAUSA300_2164 and SAUSA300_0883 have not been studied, but their heparin binding ability suggests that they probably increase cell-cell interaction by binding to heparin.

Detection of a few CWA proteins in the secreted fraction could be due to cell lysis. However, CWA proteins were not detected in the cell wall fraction. It is plausible that the large size (>100 kDa) might prevent the proteins from being captured in the heparin-binding assay. Moreover, CWA proteins have been reported to be low during planktonic growth compared to the immature biofilms (47). Also, high protease activity during the stationary phase of growth would likely result in their cleavage and localization in the secreted fraction than in the cell wall fraction.

Five cytoplasmic proteins were among the top 10 heparin-binding proteins in both the secreted and cell wall fractions: enolase, elongation factor Ts, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor Tu, and a Zn-dependent alcohol dehydrogenase (Table 2). Although cytoplasmic proteins in the cell wall fraction could be the result of contamination, recent studies suggest that this is not the case. Cytoplasmic proteins such as GAPDH and enolase have been reported to be a main constituent of the S. aureus biofilm matrix (38). These proteins reversibly associate with the cell surface at low pH and interact with eDNA to form a network of cells within the matrix (48). Additionally, nucleic acid-binding proteins such as IsaB (49), HU, elongation factors, and ribosomal proteins were detected in the secreted and cell wall fractions, consistent with the hypothesis that heparin can substitute for eDNA in the biofilm matrix.

There are some reports of toxins contributing to cell-cell interactions. The pore-forming cytotoxin alpha-hemolysin (Hla) has a role in S. aureus biofilm formation (50) and was identified in high levels of the secreted fraction (Table 2). Additional heparin-binding toxins included Panton-Valentine leukocidin (LukSF), enterotoxin Q, and LukG. The signal peptide of LukSF has been reported to act as a bridge between S. aureus and the heparin/heparan sulfate component of host extracellular matrix (ECM) in vivo (51). Although this signal peptide was not detected specifically in the heparin-binding assay, it is likely that the processed proteins possess a heparin-binding property as well.

The appearance of multiple proteins with diverse physiological roles in the heparin-bound fraction suggests that the interaction between heparin and protein is somewhat nonspecific. However, heparin-binding motifs have been identified in some proteins from higher organisms, and these motifs consist of a linear array of basic and hydrophobic amino acids. Accordingly, the heparin-binding proteins identified here were scanned for three well-characterized heparin-binding motifs (HBMs) that have been identified in eukaryotic proteins, namely, the Cardin, Weintraub, and Sobel motifs (52, 53). Approximately 50% of the heparin-bound proteins from S. aureus possessed one or more of these HBMs (Table 2). The absence of obvious HBMs in many staphylococcal proteins with heparin-binding ability suggests that they may contain motifs different from previously defined eukaryotic HBMs.

Analysis of mutants lacking individual heparin-binding proteins for their ability to form heparin-dependent biofilms.Seventeen of the 40 heparin-binding proteins identified from the secreted fraction were tested for their significance in follow-up biofilm assays using insertion mutants from the Nebraska Transposon Mutant Library (15). These 17 mutants were chosen because they are known or predicted to be either secreted or surface exposed, and transposon mutants were available in the library. All 17 of the mutants still responded to heparin by forming an enhanced biofilm, but in several cases, the magnitude of the response was relatively modest (Table 3). For example, whereas exogenous heparin increased biofilm biomass of the wild-type LAC strain by 80 to 100%, more modest increases of 20 to 40% were observed from mutants lacking IsaB, N-acetylmuramoyl-l-alanine amidase, Panton-Valentine leukocidin, LukS-PV, and a leukocidin/hemolysin toxin family protein (SAUSA300_1974) (Table 3). The fact that none of the mutants assayed completely lost the ability to respond to heparin suggests that there is redundancy in the binding ability. Additionally, essential cytoplasmic proteins such as GAPDH and enolase, whose mutants were not available in the transposon library, might play an important role in heparin-containing biofilms.