New Results

Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

Perrin Baker, Preston J. Hill, Brendan D. Snarr, Noor Alnabelseya, Mathew J. Pestrak, Mark J. Lee, Laura K. Jennings, John Tam, Roman Melnyk, Matthew R. Parsek, Donald C. Sheppard, Daniel J. Wozniak, P. Lynne Howell
doi: https://doi.org/10.1101/032714

Abstract

Bacterial biofilms are a significant medical challenge as they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases – PelAh and PslGh – encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix and that nanomolar concentrations of these enzymes can both prevent biofilm formation as well as rapidly disrupt preexisting biofilms in vitro. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58–94%. These non-cytotoxic enzymes potentiated antibiotics as the addition of either enzyme to a sub-lethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude. Additionally, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases and synthetic biology to develop novel anti-biofilm therapeutics.

Introduction

Bacterial biofilms provide a protective lifestyle for bacteria and are extremely challenging and costly to treat, as they are notoriously recalcitrant to antibiotics and host defenses (1–5). It is estimated that 65–80% of all human bacterial infections are biofilm related (6). Biofilms are complex communities of bacteria embedded in a extracellular matrix composed of proteins, extracellular DNA (eDNA) and exopolysaccharides. The exopolysaccharide component of biofilms can function to impair antibiotic penetration (7, 8) and provide a barrier against phagocytosis by host immune cells (9). Given the rise of antibiotic resistance and the discovery that sub-inhibitory concentrations of antibiotics and antimicrobial compounds can promote biofilm formation, there is an urgent need for novel and effective treatments that target and disrupt biofilms (10–12). While several compounds have been demonstrated to inhibit Pseudomonas aeruginosa biofilm formation (13–22), most are unable to disrupt established biofilms, which is more clinically relevant.

P. aeruginosa is a ubiquitous, Gram-negative, opportunistic pathogen that is commonly associated with nosocomial infections (23). Mortality associated with P. aeruginosa infections is high (24), and the emergence of multi- and even pan-resistance to antimicrobials have been reported (25). The bacterium has the genetic capacity to synthesize at least three different biofilm exopolysaccharides: Psl, Pel and alginate. These polysaccharides are integral components of the extracellular biofilm matrix (26, 27). Although alginate production results in a mucoid phenotype and is correlated with chronic infection and poor prognosis in patients with cystic fibrosis, this exopolysaccharide is dispensable for biofilm formation in non-mucoid P. aeruginosa strains (28–30). Psl is a neutral polysaccharide composed of a pentasaccharide repeat unit of D-mannose, L-rhamnose and D-glucose (31) while Pel has been recently identified as a cationic polysaccharide composed of partially acetylated N-acetyl-D-glucosamine and N-acetyl-D-galactosamine (32, 33). Psl, and under some circumstances, Pel, function to facilitate initial surface attachment (31, 32, 34–36). Both exopolysaccharides play a significant role in the formation and maintenance of the biofilm architecture (7, 37, 38). P. aeruginosa strains with genetic deletions of the pel and psl operons are profoundly impaired in biofilm formation and virulence in a mouse model of acute infection (29). Psl provides protection against neutrophil phagocytosis and antibiotics with diverse biochemical properties (8, 9) while Pel enhances resistance to aminoglycosides (7, 39). While the preference of Pel or Psl is often strain specific, many isolates are capable of switching between the synthesis of Pel and Psl in response to stress to maintain infection in the host (40, 41). This adaptive mechanism underscores the importance of developing therapies that target both exopolysaccharides.

The utilization of Pel and Psl for antibiotic resistance and virulence makes them attractive targets for the prevention and treatment of P. aeruginosa infection. However, compounds that target and disrupt these integral biofilm components have not been identified. We therefore sought to identify enzymes that selectively target and degrade Psl and Pel. One common feature shared among many exopolysaccharide biosynthetic operons is the presence of a gene encoding a glycoside hydrolase that is proposed to hydrolyze the exopolysaccharide produced by the biosynthetic pathway (33, 42–46). We have exploited these naturally derived glycoside hydrolases as a method of biofilm prevention and dispersal. We demonstrate that the addition of low nanomolar concentrations of these enzymes – PelAh and PslGh, – can both prevent biofilm formation as well as disrupt existing biofilms of laboratory, clinical and environmental isolates of P. aeruginosa in vitro at nanomolar concentrations. In addition to disrupting biofilms, these non-cytotoxic enzymes potentiate antibiotics and enhance susceptibility to killing by neutrophils. These studies provide us with a method to find enzymes with anti-biofilm activity for the treatment and eradication of chronic bacterial infections.

Results

Design and Expression of Enzymatically Active Glycoside Hydrolases. The Gramnegative bacterium Actinobacillus actinomycetemcomitans secretes a biofilm-modifying glycoside hydrolase that allows for the dispersal from the biofilm (47), while the Grampositive Bacillus licheniformis secrets nucleases to disrupt established biofilms and inhibit biofilm formation of competing bacteria (48). To date, no secreted biofilm dispersal enzymes have been identified in P. aeruginosa. However, previous bioinformatics analyses have identified PslG and the N-terminal domain of PelA as putative periplasmic glycoside hydrolases encoded in the psl and pel biosynthetic operons, respectively (33). We recently purified and functionally characterized PslG31–442, a member of glycoside hydrolase family 39 herein referred to as PslGh. This construct removes an N-terminal transmembrane domain, producing a soluble, catalytically active, glycoside hydrolase domain that can hydrolyze Psl (49). PelA is a bifunctional protein that contains at least two catalytic domains, a putative glycoside hydrolase domain and a CE4 deacetylase domain (33). Based on bioinformatics prediction with the CAZymes Analysis Toolkit (50), we generated a PelA47–303 construct, herein referred to as PelAh, to explicitly study the activity of the glycoside hydrolase domain. This construct was soluble and could be purified to homogeneity using Ni-NTA purification and size-exclusion chromatography, yielding 50 mg of protein per liter of bacterial cell culture.

Enzyme-Catalyzed Disruption of Biofilms. We hypothesized that the exogenous application of the glycoside hydrolases PelAh and PslGh to Pel and Psl dependent biofilms, respectively, would result hydrolysis of the exopolysaccharides, thereby disrupting these established biofilms. To assay for biofilm disruption, biofilms were produced using the following strains: PA14 (Pel dependent matrix), PAO1 (Psl dependent matrix), and L-arabinose inducible P. aeruginosa PAO1 ΔwspF Δpsl PBADpel, and PAO1 ΔpelF PBADpsl, which exclusively produce Pel and Psl, respectively. PelAh, and a putative catalytically inactive E218A variant (PelAh E218A) were applied to Pel-dependent biofilms, while PslGh and an inactive E165Q/E276Q variant (PslGh E165Q/E276Q) were applied to Psl-dependent biofilms. Confocal microscopy coupled with fluorescently-labeled lectin staining with lectins specific for Psl (Hippeastrum hybrid, HHA) and Pel (Wisteria Floribunda, WFL) demonstrated that catalytically active hydrolases, but not the inactive variants, were capable of degrading the Pel-dependent and Psl-dependent biofilm biomass based on the elimination of fluorescent signal following treatment (Fig 1).

Figure 1.
Figure 1.

The glycoside hydrolases PslGh and PelAh hydrolyze the exopolysaccharides Pel and Psl in a biofilm. Representative confocal images of Psl biofilms grown statically for 24 h (Top) and Pel biofilms cultivated for 48 h (Bottom) under flow conditions and treated with the indicated hydrolases. Biofilms were stained with the HHA Psl-specific lectin (green) and WFL Pelspecific lectin (red) (Scale bars: 30 μm).

Crystal violet staining was subsequently utilized to quantify the effect of hydrolase treatment on the total biofilm biomass. A 2 h treatment of a Pel-dependent biofilm with PelAh resulted in disruption of 99% of the biomass, while both the PelAh E218A variant and PslGh, added in 100-fold excess compared to PelAh, exhibited no significant difference compared to that of the untreated biofilm (Fig 2A). Similar results were obtained for Psl-dependent biofilms wherein only treatment with a catalytically active PslGh resulted in a 98.5% reduction in biofilm biomass. The activity of both enzymes was observed to be dose dependent. When incubated with established biofilms for 1 h, PelAh exhibited an EC50 of 35.7 ± 1.1 nM, while PslGh had an EC50 of 12.9 ± 1.1 nM (Fig 2B). Time course experiments using fixed concentrations of PelAh and PslGh revealed a continuous decrease in biofilm biomass over time (Fig S1). Combined, these results indicate that biofilm-dispersal is catalytic, rapid, and exopolysaccharide specific.

Figure S1.
Figure S1.

Time course disruption of P. aeruginosa biofilms. Crystal violet staining of biofilms following the exogenous addition of glycoside hydrolases (A) PelAh and (B) PslGh on their respective exopolysaccharide. Each data point represents the mean from n = 3 crystal violet microtiter plate wells. Error bars indicate SEM.

Figure 2.
Figure 2.

P. aeruginosa biofilm inhibition and disruption by glycoside hydrolases (A) Crystal violet staining of biofilms following the exogenous addition of glycoside hydrolases or catalytic variants. (B) Dose-response curves to examine the dispersal of biofilm biomass by the exogenous treatment of each glycoside hydrolase and variant. (C) Dose-response curves to examine the prevention of biofilm biomass in the presence of various glycoside hydrolases. Each data point represents the mean from three independent experiments of n = 3 crystal violet microtiter plate wells. Error bars indicate SEM. The *** indicates a significant difference (p ≤ 0.001) and NS represents no significant difference.

Glycoside Hydrolases Inhibit Biofilm Formation but not Bacterial Growth. Since the glycoside hydrolases were effective at disrupting established biofilms, we next sought to determine whether the application of enzyme to bacterial culture could be utilized as a prophylactic strategy to prevent biofilm formation. The addition of PelAh, but not PslGh, to Pel-producing P. aeruginosa abrogated biofilm formation. A dose-titration indicated that Pel-biofilms could be prevented over 24 h by PelAh addition with an EC50 of 69.3 ± 1.2 nM (Fig 2C). As visualized in borosilicate tubes, PelAh prevented pellicle biofilm at the air-liquid interface and bacterial cells grew exclusively in the planktonic state (Fig S2). Interestingly, addition of PelAh E218A, which cannot catalyze the dispersal of biofilms, resulted in a statistically significant reduction in biofilm biomass at concentrations ≥500 nM. While an accurate EC50 value could not be readily determined for this catalytic variant, 5 μM of the enzyme variant (>70 times greater than the EC50 of the wild-type) resulted in <50% reduction of the biomass relative to untreated cells. The presence of ≥10 μM PslGh did not affect the ability of P. aeruginosa to form Pel-dependent biofilms. Consistent with results for PelAh on Pel-dependent biofilms, the addition of 1 μM PslGh to Psl producing cultures under biofilm forming conditions resulted in a complete inhibition of biofilm formation, and a dosetitration indicated that PslGh had an EC50 of 4.1 ± 1.1 nM over 24 h. To examine whether the effect was the direct result of PslGh activity, the catalytically inactive variant PslGh E165Q/E276Q was tested. This variant was >100-fold less effective at biofilm prevention (EC50 of 466.5 ± 1.1 nM) relative to the catalytically active enzyme. Addition of ≥10 μM PelAh, had no affect on Psl-dependent biofilm production.

Figure S2.
Figure S2.

Biofilm prevention standing culture pellicle assay. Biofilm formation at the airliquid interface was examined in Pel-dependent culture following incubation with PelAh and PslGh. Arrows indicate the location of the air-liquid interface where biofilm formation occurs.

We next examined the length of time that the glycoside hydrolases could prevent biofilm formation. A single dose of PelAh or PslGh prevented biofilm formation for 48 and 72 h, respectively. The formation of a Pel-dependent biofilm at 72 h was associated with proteolytic degradation of PelAh, while no degradation of PslGh was observed over the entirety of the experiment (Fig S3). The growth rate of P. aeruginosa PAO1 exposed to ≥20 μM of either glycoside hydrolase was unaffected over six hours of static growth when compared to a no treatment control (Fig S4). The absence of enzyme cross-reactivity between exopolysaccharides indicates that biofilm inhibition is highly specific. This result, combined with bacterial growth curves, demonstrates that exogenous glycoside hydrolases do not impede biofilm formation by altering cell viability and growth.

Figure S3.
Figure S3.

Protein stability of PelAh and PslGh in P. aeruginosa culture. Western blotting using α-PelA and α-PslG to detect the presence of exogenously applied PelAh and PslGh at various time points during incubating with P. aeruginosa Pel and Psl biofilm formation, respectively. Incubation of each glycoside hydrolase in the absence of P. aeruginosa culture (Cell -) was utilized for comparison.

Figure S4.
Figure S4.

Glycoside hydrolases do not affect P. aeruginosa growth. Growth curve with P. aeruginosa PAO1 in the presence of PslGh and PelAh over 6 h at 37 °C.

Biofilm-Dispersing Enzymes are Non-Cytotoxic. Since exogenous PelAh and PslGh did not affect P. aeruginosa cell viability and growth, we next sought to examine whether enzyme treatment impacted mammalian cells. IMR-90 human lung fibroblast cells treated for 5 h with concentrations up to 1 mg/mL of either enzyme, ~100-fold above the concentration required for effective biofilm disruption, resulted in no significant difference in cell area or length-to-width ratio (Fig 3A). Following 48 h incubation, no significant difference in cellular viability was observed in PelAh and PslGh treated cells regardless of the media used (Fig 3B). Clostridium difficile toxin TcdB (51) was used as a control for cell morphology as it results in cell rounding, while digitonin, which permeabilizes the cells, was used to monitor cellular viability (Fig 3B). Western blotting of the media confirmed that PelAh and PslGh remained intact for the 48 h duration of the experiment (Fig S5). Together, these results suggest that the enzymes do not interfere with mammalian cell morphology and viability.

Figure S5.
Figure S5.

Protein stability of PelAh and PslGh in mammalian cell culture. Western blotting of exogenously added PelAh and PslGh after 48 h incubation in IMR-90 cell culture in the presence and absence of 10% FBS in the media.

Figure 3.
Figure 3.

The glycoside hydrolases PelAh and PslGh are non-cytotoxic. (A) IMR-90 cellomics assay to measure the length-to-width ratio (LWR) of the cells using CellTracker Orange CMRA. (B) IMR-90 fibroblast cell viability assay using PrestoBlue® reagent. All data was normalized to a no treatment control (100%). The C. difficle toxin TcdB was used as a positive control in cell morphology assays and the detergent digitonin was utilized as a negative control in cell viability assays. Each data point represents the mean from three independent experiments of n = 3 from cellomic and PrestoBlue® measurements in microtiter plate well. Error bars indicate SEM. The *** indicates a significant difference (p ≤ 0.001) and NS represents no significant difference.

Enzymes Potentiate Antibiotics and Ameliorate Human Neutrophil Killing. Previous studies have demonstrated that both Pel and Psl enhance antibiotic resistance (7, 8). We therefore theorized that glycoside hydrolase degradation of these polymers could potentiate the activity of antimicrobial agents. As the antibiotic colistin targets the cell membrane of P. aeruginosa, it is active against both the metabolically active and dormant cells found within biofilms (52). We therefore examined the effect of combining glycoside hydrolase treatment with sub-inhibitory concentrations of colistin. As predicted, treatment of P. aeruginosa Pel- and Psl-dependent biofilm cultures with colistin (50 μg/mL), PelAh, or PslGh (2 μM) alone had no effect on the viability of the bacteria (Fig 4A). However, the combination of PelAh or PslGh with colistin resulted in a ~2.5 log reduction in bacterial colony forming units. This result indicates that these glycoside hydrolases are compatible with antibiotics and can potentiate the antimicrobial activity of colistin.

Figure 4.
Figure 4.

Glycoside hydrolases potentiate antibiotics and increase human neutrophil killing. (A) Cell number count for PAO1 ΔwspF Δpsl PBAD pel (Left) and PAO1 ΔpelF PBAD psl (Right) and in the presence of glycoside hydrolases, antibiotic and a combined enzyme + antibiotic treatment. Mean was calculated from LB agar plate counts from 3 independent experiments. (B) HL60 neutrophil killing of strain PAO1 ΔwspF Δpsl PBAD pel and PAO1 ΔpelF PBAD psl following biofilm formation and treatment with PelAh and PslGh and their catalytic variants, respectively. Percent killing was normalized to a no treatment control. Error bars indicate SEM. The * indicates a significant difference of (p ≤ 0.05), *** indicates (p ≤ 0.001) and NS represents no significant difference.

Since P. aeruginosa exopolysaccharide enhance resistance to human neutrophil killing, we next investigated whether glycoside hydrolase treatment could enhance susceptibility to immune-mediated killing. It has been demonstrated in vitro that exopolysaccharide produced by P. aeruginosa enhance resistance to neutrophils by inhibiting efficient opsonization and reducing reactive oxygen species production (9). To determine if hydrolase treatment could impact neutrophil killing, the ability of PelAh and PslGh to enhance the susceptibility of P. aeruginosa to the human HL-60 neutrophil cell line was examined. Treatment of Pel-containing P. aeruginosa biofilms with PelAh increased the degree of HL-60-mediated microbial killing from approximately 22% to 42% (Fig 4B). This was not observed in a PelAh E218A variant indicating that the enhanced susceptibility to neutrophils is due to the catalytic activity of the enzyme that disrupts the biofilm. This effect was specific to PelAh, as neither PslGh nor the E165Q/E276Q variant were significant to ameliorate neutrophil killing, or affect neutrophil activity (Fig 4B). Combined, these data provide further evidence that PelAh and PslGh do not affect mammalian cell function, and that PelAh can function to enhance neutrophil killing of P. aeruginosa.

Enzymes effectively Disrupt Biofilms from Clinical and Environmental Isolates. Our previous work established that clinical strains of P. aeruginosa can be divided into four different classes based upon their dependence on Pel and Psl exopolysaccharides for biofilm formation (40). Biofilm production in Class I strains is dependent on Pel, Class II strains are Psl dependent, Class III strains are redundant exopolysaccharide matrix producers and Class IV strains are matrix over-producers. In Class III and IV, both Pel and Psl contribute to biofilm formation. The effect of PelAh and PslGh on biofilm dispersal of isolates from each of the four classes was evaluated. Consistent with results previously reported, Class III environmental isolates exhibited poor attachment and biofilm formation while the cystic fibrosis clinical isolate CF127, a Class IV strain, produced the greatest biomass (40). Under the growth and assay conditions tested, treatment with 300 nM PslGh +PelAh for 2 h resulted in a 70–94% dispersal of biofilms formed by isolates from classes II-IV (Fig 5). PslGh was more effective at disrupting biofilms from classes II-IV, suggesting that the Psl polysaccharide is a major contributor to biofilm biomass. An additive effect was observed for the matrix-overproducer CF127 where the combination of both enzymes led to the largest decrease in biofilm biomass (95% biomass reduction for PslGh+PelAh compared to 85% for PslGh alone). Strains PA14, the sole member of Class I and class IV member CF127 required 1 μM of enzyme to reduce the biofilm biomass by 58% and 95%, respectively. These data demonstrates that the enzymes are compatible with one another and can be utilized to disrupt biofilms of clinical and environmental P. aeruginosa isolates.

Figure 5.
Figure 5.

Enzymatic biofilm dispersal of P. aeruginosa clinical and environmental isolates. Isolates were grouped into categories as previously described and glycoside hydrolases PelAh and PslGh were exogenously added either individually or together and allowed to incubate for 2 h. Each data point represents the mean from three independent experiments of n = 3 crystal violet microtiter plate wells. Error bars indicate SEM. The *** indicates a significant difference (p ≤ 0.001).

Discussion

In this work, we demonstrate that the glycoside hydrolases, PslGh and PelAh, encoded in the exopolysaccharide biosynthetic operons of P. aeruginosa can be utilized to prevent and disrupt biofilms produced by the bacterium. We establish that these non-cytotoxic enzymes potentiate antibiotics and the innate immune system suggesting that they are promising enzymes for the treatment of P. aeruginosa infections. Since many Gram -positive and - negative bacterial exopolysaccharide biosynthetic operons encode a putative glycoside hydrolase or lyase, it is likely that this strategy could be extended to prevent and disrupt other exopolysaccharide-dependent biofilms. Examples include BcsZ, WssD, PssZ and PgaB involved in cellulose, acetylated cellulose, Listeria monocytogenes exopolysaccharide and poly-β-1,6-N-acetyl-D-glucosamine (PNAG) biosynthesis, respectively (42, 43, 45, 46). Glycoside hydrolase therapy therefore has the potential to target many Gram -positive and - negative biofilms from bacteria that are extremely relevant in healthcare and industrial settings.

Despite aggressive antimicrobial therapy, P. aeruginosa colonization is rarely eradicated and can lead to devastating biofilm-related persistent infections. Since exopolysaccharides that comprise the P. aeruginosa biofilm matrix aid in resistance to antimicrobial agents (7, 8, 39, 53), studies have focused on inhibiting biofilm formation through the activation of bacterial response mechanisms. Halogenated furanones and their derivatives (13–15), cysteine sulfoxide compounds, (16), 6-gingerol (17) and meta-bromo-thiolactone (18) all target quorum-sensing signals to prevent P. aeruginosa biofilm formation. The use of Damino acids, which alter the cell envelope (19), and antimicrobial peptides (AMP) (20–22) which are capable of penetrating the biofilm have also been explored. Disappointingly, only a few molecules, including: nitric oxide (54), the fatty messenger cis-2-decenoic acid (CDA) (55) and the AMP 1080 (22), mediate both P. aeruginosa biofilm prevention and disruption. Nitric oxide decreases levels of the secondary messenger c-di-GMP levels, thereby down-regulating exopolysaccharide production and promoting planktonic growth (56), while CDA up-regulates genes involved in motility and metabolic activity and down-regulates genes contributing to attachment (57). Unfortunately, these molecules have only been tested on P. aeruginosa PAO1, and as the activity of these agents may not be specific to P. aeruginosa, they may negatively affect the natural microbiota. With the exception of cis-2-decenoic acid, these agents also require extended incubation times (≥ 24 h) to be efficacious against established biofilms. Therapeutic small molecules targeting cellular processes within the bacterial cytosol face numerous barriers including the biofilm matrix and two membranes that result in attrition. During this process many compounds are susceptible to multiple microbial resistance mechanisms including; impermeability of the membrane, sequestration, overproduction of the target, enzyme modification and multi-drug efflux.

Enzymes as therapeutics exhibit three inherent features that distinguish them from traditional small molecule drugs in that they bind and act on their targets with high affinity and specificity, their catalytic nature allows for the conversion of multiple target molecules, and because they target extracellular processes are prone to fewer resistance mechanisms. Therefore, the use of biofilm-dispersing enzymes has become recognized as a promising therapeutic strategy for the prevention and disruption of bacterial biofilms over the sole use of small molecule inhibitors, antibiotics and AMPs (58–60). DspB (DispersinB®), a glycoside hydrolase produced by Actinobacillus actinomycetemcomitans, hydrolyzes PNAG, a important exopolysaccharide required for biofilm formation and integrity by several Gram - positive and -negative pathogenic bacteria, but not P. aeruginosa (47, 48, 61–64). Dornase alfa (DNase I), which is in clinical use, disrupts bacterial biofilms through the hydrolysis of eDNA, hence reducing biofilm biomass and potentiating some antibiotics (65, 66). While young P. aeruginosa biofilms are sensitive to DNaseI treatment based on the role of eDNA during initial biofilm establishment, mature biofilms are less susceptible presumably since the exopolysaccharide changes during development (67, 68). Our results indicate that PslGh and PelAh are compatible with antibiotics and neutrophils, and function to rapidly and selectively target exopolysaccharide on the bacterial surface. This mechanism of action reduces susceptibility to host resistance mechanisms and off-target effects to the microbiota and host carbohydrates based upon the unique chemical structures of Pel and Psl. While the exopolysaccharide binds biofilm matrix components including eDNA and proteins to form a cohesive and structurally robust biofilm (69, 70), both glycoside hydrolases are able to access and hydrolyze the polysaccharides. Penetration and dispersal of the biofilm has several potential consequences including; increased penetration of antibiotics within the biofilm matrix, and the reduction of microenvironments that can render antimicrobials inactive. Since many infections are often polymicrobial, electing to target a single bacterial species may be perceived as inefficient. However, Pel has been demonstrated to promote mixed species biofilms with Staphylococcus aureus (71), and Psl-mediated protection against detergent stress and antibiotics is conferred to other pathogen bacteria in mixed species biofilms (8, 72). Therefore, hydrolysis of Pel and Psl in mixed species biofilms could increase the susceptibility of other embedded pathogenic bacteria to conventional therapeutic strategies.

In conclusion, bacterial biofilms are notoriously recalcitrant to immune defences and antibiotics and therefore novel therapeutic methods are urgently needed. Our study demonstrates that components of the P. aeruginosa exopolysaccharide biosynthetic operons can be manipulated to disrupt these biofilms allowing for antibiotic potentiation and effective killing by innate immunity. We hypothesize that glycoside hydrolases from other exopolysaccharide biosynthetic pathways can also be exploited to target diverse biofilms in a wide-range of applications.

Materials and Methods

Strains and culture conditions. Strains used in this study are detailed in Table S1 and detailed culture conditions are described in SI Materials and Methods.

View this table:
Table S1:

Strains and Plasmids used in this Study.

Cloning, Expression, and Purification of PelA and PslG constructs. PslGh was purified as previously described (49). The DNA sequence of pelA from P. aeruginosa PAO1 was obtained from GenBank under accession no. AAG06452.1 (73). The PRED-TAT server (74) indicates that PelA possesses a TAT signal sequence from residues 1–45. Full experimental detail for cloning, expression and purification of PelAh are provided in the SI Materials and Methods.

Confocal Microscopy, Mitrotiter Dish Biofilm Assays and P. aeruginosa growth assays.

Full experimental detail for confocal microscopy, mitrotiter dish biofilm assay and P. aeruginosa assays can be found in SI Materials and Methods.

Antibiotic sensitivity assay. Overnight cultures of P. aeruginosa PAO1 ΔwspF Δpsl PBADpel (Pel-dependent) and P. aeruginosa PAO1 ΔpelF PBADpsl (Psl-dependent) were diluted to an OD600 of 0.05 in LB no salt (LBNS) with 0.5% arabinose and grown statically in polystyrene tubes in a final volume of 1 mL/tube. PelAh and PslGh were added at a final concentration of 2 μM and incubated with exopolysaccharide producing cells for 24 h at 25°C. Cultures which did not contain enzyme served as untreated samples. Following incubation, colistin was added to all tubes at a final concentration of 50 μg/mL and incubated for 24 h. Adherent biofilm and embedded cells were resuspended by scraping the tubes and vigorous pipetting to ensure removal of all cellular material and prevent experimental bias. Viability was quantified by serial dilutions and CFU counts on LB agar plates of the surviving population. Experiments were performed three times to obtain an average and standard error.

Human neutrophil killing assay. Overnight cultures of P. aeruginosa PAO1 ΔwspF Δpsl PBADpel and P. aeruginosa PAO1 ΔpelF PBADpsl were diluted to an OD600 of 0.05 in LBNS with 0.5% L-arabinose and inoculated in a 96 well tissue culture-treated plate at a final volume of 100 μL/well. The plate was incubated statically at 28°C for 20 h. Supernatants were aspirated and 100 μL of phenol red-free Roswell Park Memorial Institute media (RPMI)+10% FBS containing 0.5 μM of PelAh or PslGh was added. The plate was incubated at room temperature on a Nutator for 1 h. Following pretreatment with hydrolase, 100 μL RPMI+10% FBS containing 6×106 differentiated HL-60 cells were added to the wells, and plate was incubated for 90 min at 37 °C, 5% CO2. Wells were aspirated and supernatant was diluted between 1/200000 and 1/400000, and plated (50 μL) onto LB agar. To aspirated wells, 200 μL of 2 μM of PelAh and 2 μM PslGh was added and plate was incubated at room temperature on the Nutator for 1–1.5 h. Wells were aspirated, diluted and plated onto LB agar as above.

Cell morphology and viability assays. IMR-90 human lung fibroblast cells were seeded in 96-well CellBIND plates (Corning®) at a density of 8,000–10,000 cells/well. The next day, the media was exchanged with media containing 1 μM CellTracker Orange CMRA (Molecular Probes) in serum-free EMEM (Wisent) media or supplemented with 10% FBS. After 60 min, excess dye was removed by media exchange. PslGh and PelAh were added to a final concentration of 1 mg/mL. The detergent digitonin and the Clostridium difficile toxin TcdB were utilized as controls and added to a final concentration of 0.03 mg/mL and 0.5 pM, respectively (51). The cell plates were returned to the incubator for 5.0 h before imaging. CellTracker-labeled cells were evaluated on a Cellomics ArrayScan VTI HCS reader (Thermo Scientific) using the target acquisition mode, a 10x objective, and a sample rate of 100 objects per well. Following 24 h incubation, cells in serum-free media were supplemented with 10% FBS. At 48 h post incubation, PrestoBlue® reagent was added at a 1:10 reagent:media to the cells and allowed to incubate for 5.0 h. The microtiter plates were read in a SpectroMax M2 plate reader with a λx of 555 nm and λem of 585 nm. Western blotting to probe for the presence of PslGh and PelAh was completed using polyclonal antibodies that were previous generated against both enzymes (33, 49).

Statistical Analysis. One-way ANOVA and Tukey’s multiple comparison test were utilized to valuate statistical significance. EC50 values were calculated using non-linear least-squares fitting to a dose-response model in Prism (GraphPad Software) All means, SEM, bar graphs and dose-response curves were calculated and generated using Prism 6.0.

ACKNOWLEDGEMENTS

*Research described in this paper is supported by operating grants from the Canadian Institutes of Health Research (CIHR) (#43998 to P.L.H., #123306 to D.C.S., #286650 to R.A.M., and #81361 to P.L.H. and D.C.S.), Cystic Fibrosis Canada (CFC) (D.C.S. and P.L.H.), the National Institutes of Health (R01AI097511 to D.J.W. and 2R01AI077628 to M.R.P.) and the Natural Sciences and Engineering Research Council of Canada (RGPIN 418405 to R.A.M.). P.B. has been supported in part by a CFC postdoctoral fellowship and a Banting Fellowship from CIHR. B.D.S has been supported by graduate scholarships from CFC and CIHR. L.K.J. is the recipient of an American Heart Association Postdoctoral Fellowship (14POST20130017). P.L.H is the recipient of a Canada Research Chair.

Footnotes

  • ↵* Research described in this paper is supported by operating grants from the Canadian Institutes of Health Research (CIHR) (#43998 to P.L.H., #123306 to D.C.S., #286650 to R.A.M., and #81361 to P.L.H. and D.C.S.), Cystic Fibrosis Canada (CFC) (D.C.S. and P.L.H.), the National Institutes of Health (R01AI097511 to D.J.W. and 2R01AI077628 to M.R.P.) and the Natural Sciences and Engineering Research Council of Canada (RGPIN 418405 to R.A.M.). P.B. has been supported in part by a CFC postdoctoral fellowship and a Banting Fellowship from CIHR. B.D.S has been supported by graduate scholarships from CFC and CIHR. L.K.J. is the recipient of an American Heart Association Postdoctoral Fellowship (14POST20130017). P.L.H is the recipient of a Canada Research Chair.

REFERENCES

  1. 1.↵
    J. W. Costerton, P. S. Stewart, E. P. Greenberg, Bacterial biofilms: A common cause of persistent infections. Science 284, 1318–1322 (1999); published online EpubMay 21, 1999
    OpenUrlAbstract/FREE Full Text
  2. 2.
    C. A. Fux, J. W. Costerton, P. S. Stewart, P. Stoodley, Survival strategies of infectious biofilms. Trends Microbiology 13, 34–40 (2005).
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.
    N. Hoiby, T. Bjarnsholt, M. Givskov, S. Molin, O. Ciofu, Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Agents 35, 322–332 (2010); published online Epub04/01 (
    OpenUrlCrossRefPubMed
  4. 4.
    T. C. Mah, G. A. O’Toole, Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 9, 34–39 (2001).
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    S. L. Percival, K. E. Hill, S. Malic, D. W. Thomas, D. W. Williams, Antimicrobial tolerance and the significance of persister cells in recalcitrant chronic wound biofilms. Wound Repair Regen 19, 1–9 (2011)10.1111/j.1524–475X.2010.00651.x).
    OpenUrlCrossRefPubMedWeb of Science
  6. 6.↵
    C. Potera, Forging a link between biofilms and disease. Science 283, 1837, 1839 (1999); published online EpubMar 19 (
  7. 7.↵
    K. M. Colvin, V. D. Gordon, K. Murakami, B. R. Borlee, D. J. Wozniak, G. C. Wong, M. R. Parsek, The pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS pathogens 7, e1001264 (2011) 10.1371/journal.ppat.1001264).
    OpenUrlCrossRefPubMed
  8. 8.↵
    N. Billings, M. Millan, M. Caldara, R. Rusconi, Y. Tarasova, R. Stocker, K. Ribbeck, The extracellular matrix Component Psl provides fast-acting antibiotic defense in Pseudomonas aeruginosa biofilms. PLoSPathog 9, e1003526 (2013)10.1371/journal.ppat.1003526).
    OpenUrl
  9. 9.↵
    M. Mishra, M. S. Byrd, S. Sergeant, A. K. Azad, M. R. Parsek, L. McPhail, L. S. Schlesinger, D. J. Wozniak, Pseudomonas aeruginosa Psl polysaccharide reduces neutrophil phagocytosis and the oxidative response by limiting complement-mediated opsonization. Cellular microbiology 14, 95–106 (2012); published online EpubJan (10.1111/j. 1462-5822.2011.01704.x).
    OpenUrlCrossRefPubMed
  10. 10.↵
    Y. Morita, J. Tomida, Y. Kawamura, Responses of Pseudomonas aeruginosa to antimicrobials. Frontiers in microbiology 4, 422 (2014)10.3389/fmicb.2013.00422).
    OpenUrl
  11. 11.
    J. F. Linares, I. Gustafsson, F. Baquero, J. L. Martinez, Antibiotics as intermicrobial signaling agents instead of weapons. Proceedings of the National Academy of Sciences of the United States of America 103, 19484–19489 (2006); published online EpubDec 19 (10.1073/pnas.0608949103).
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    L. R. Hoffman, D. A. D’Argenio, M. J. MacCoss, Z. Zhang, R. A. Jones, S. I. Miller, Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436, 1171–1175 (2005); published online EpubAug 25 (10.1038/nature03912).
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    M. Hentzer, M. Givskov, Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections. The Journal of clinical investigation 112, 1300–1307 (2003); published online EpubNov (10.1172/JCI20074).
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.
    M. Hentzer, K. Riedel, T. B. Rasmussen, A. Heydorn, J. B. Andersen, M. R. Parsek, S. A. Rice, L. Eberl, S. Molin, N. Hoiby, S. Kjelleberg, M. Givskov, Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148, 87–102 (2002); published online EpubJan (
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    C. Kim, J. Kim, H. Y. Park, H. J. Park, J. H. Lee, C. K. Kim, J. Yoon, Furanone derivatives as quorum-sensing antagonists of Pseudomonas aeruginosa. Applied microbiology and biotechnology 80, 37–47 (2008); published online EpubAug (10.1007/s00253-008-1474-6).
    OpenUrlCrossRefPubMed
  16. 16.↵
    N. C. Cady, K. A. McKean, J. Behnke, R. Kubec, A. P. Mosier, S. H. Kasper, D. S. Burz, R. A. Musah, Inhibition of biofilm formation, quorum sensing and infection in Pseudomonas aeruginosa by natural products-inspired organosulfur compounds. PloS one 7, e38492 (2012) 10.1371/journal.pone.0038492).
    OpenUrlCrossRefPubMed
  17. 17.↵
    H. S. Kim, S. H. Lee, Y. Byun, H. D. Park, 6-Gingerol reduces Pseudomonas aeruginosa biofilm formation and virulence via quorum sensing inhibition. Scientific reports 5, 8656 (2015)10.1038/srep08656).
    OpenUrl
  18. 18.↵
    C. T. O’Loughlin, L. C. Miller, A. Siryaporn, K. Drescher, M. F. Semmelhack, B. L. Bassler, A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation. Proceedings of the National Academy of Sciences of the United States of America 110, 17981–17986 (2013); published online EpubOct 29 (10.1073/pnas.1316981110).
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    I. Kolodkin-Gal, D. Romero, S. Cao, J. Clardy, R. Kolter, R. Losick, D-amino acids trigger biofilm disassembly. Science 328, 627–629 (2010); published online EpubApr 30 (10.1126/science. 1188628).
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    S. Dosler, E. Karaaslan, Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides. Peptides 62, 32–37 (2014); published online EpubDec (10.1016/j.peptides.2014.09.021).
    OpenUrlCrossRefPubMed
  21. 21.
    J. Overhage, A. Campisano, M. Bains, E. C. Torfs, B. H. Rehm, R. E. Hancock, Human host defense peptide LL-37 prevents bacterial biofilm formation. Infection and immunity 76, 41764182 (2008); published online EpubSep (10.1128/IAI.00318–08).
    OpenUrl
  22. 22.↵
    C. de la Fuente-Nunez, F. Reffuveille, E. F. Haney, S. K. Straus, R. E. Hancock, Broad-spectrum anti-biofilm peptide that targets a cellular stress response. PLoS pathogens 10, e1004152 (2014); published online EpubMay (10.1371/journal.ppat.1004152).
    OpenUrlCrossRefPubMed
  23. 23.↵
    M. D. Obritsch, D. N. Fish, R. MacLaren, R. Jung, Nosocomial infections due to multidrug-resistant Pseudomonas aeruginosa: epidemiology and treatment options. Pharmacotherapy 25, 1353–1364 (2005); published online EpubOct (10.1592/phco.2005.25.10.1353).
    OpenUrlCrossRefPubMedWeb of Science
  24. 24.↵
    C. I. Kang, S. H. Kim, H. B. Kim, S. W. Park, Y. J. Choe, M. D. Oh, E. C. Kim, K. W. Choe, Pseudomonas aeruginosa bacteremia: risk factors for mortality and influence of delayed receipt of effective antimicrobial therapy on clinical outcome. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 37, 745–751 (2003); published online EpubSep 15 (10.1086/377200).
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    D. M. Livermore, Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare? Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 34, 634–640 (2002); published online EpubMar 1 (10.1086/338782).
    OpenUrlCrossRefPubMedWeb of Science
  26. 26.↵
    H. C. Flemming, J. Wingender, The biofilm matrix. Nat Rev Micro 8, 623–633 (2010).
    OpenUrl
  27. 27.↵
    E. E. Mann, D. J. Wozniak, Pseudomonas biofilm matrix composition and niche biology. FEMSMicrobiol.Rev. 36, 893–916 (2011)10.1111/j.1574–6976.2011.00322.x).
    OpenUrl
  28. 28.↵
    D. J. Wozniak, T. J. Wyckoff, M. Starkey, R. Keyser, P. Azadi, G. A. O’Toole, M. R. Parsek, Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms. Proceedings of the National Academy of Sciences of the United States of America 100, 7907–7912 (2003); published online Epub Jun 24 (10.1073/pnas. 1231792100).
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    L. Yang, W. Hengzhuang, H. Wu, S. Damkiaer, N. Jochumsen, Z. Song, M. Givskov, N. Hoiby, S. Molin, Polysaccharides serve as scaffold of biofilms formed by mucoid Pseudomonas aeruginosa. FEMS immunology and medical microbiology 65, 366–376 (2012); published online EpubJul (10.1111/j.1574–695X.2012.00936.x).
    OpenUrlCrossRefPubMed
  30. 30.↵
    A. P. Stapper, G. Narasimhan, D. E. Ohman, J. Barakat, M. Hentzer, S. Molin, A. Kharazmi, N. Hoiby, K. Mathee, Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation. Journal of medical microbiology 53, 679–690 (2004); published online EpubJul (
    OpenUrlCrossRefPubMedWeb of Science
  31. 31.↵
    M. S. Byrd, I. Sadovskaya, E. Vinogradov, H. Lu, A. B. Sprinkle, S. H. Richardson, L. Ma, Ralston, M. R. Parsek, E. M. Anderson, J. S. Lam, D. J. Wozniak, Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production. Molecular microbiology 73, 622–638 (2009); published online EpubAug (10.1111/j. 1365-2958.2009.06795.x).
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    L. K. Jennings, K. M. Storek, H. E. Ledvina, C. Coulon, L. S. Marmont, I. Sadovskaya, P. R. Secor, B. S. Tseng, M. Scian, A. Filloux, D. J. Wozniak, P. L. Howell, M. R. Parsek, Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix. Proc Natl Acad Sci U S A 112, 11353–11358 (2015); published online EpubSep 8 (10.1073/pnas.1503058112).
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    K. M. Colvin, N. Alnabelseya, P. Baker, J. C. Whitney, P. L. Howell, M. R. Parsek, PelA deacetylase activity is required for Pel polysaccharide synthesis in Pseudomonas aeruginosa. J Bacteriol 195, 2329–2339 (2013); published online EpubMay (10.1128/JB.02150–12).
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    M. S. Byrd, B. Pang, M. Mishra, W. E. Swords, D. J. Wozniak, The Pseudomonas aeruginosa exopolysaccharide Psl facilitates surface adherence and NF-kappaB activation in A549 cells. mBio 1, (2010)10.1128/mBio.00140–10).
  35. 35.
    L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, D. J. Wozniak, Analysis of Pseudomonas aeruginosa conditional psl variants reveals roles for the psl polysaccharide in adhesion and maintaining biofilm structure postattachment. J Bacteriol 188, 8213–8221 (2006); published online EpubDec (10.1128/JB.01202–06).
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    P. Vasseur, I. Vallet-Gely, C. Soscia, S. Genin, A. Filloux, The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation. Microbiology 151, 985–997 (2005); published online EpubMar (10.1099/mic.0.27410–0).
    OpenUrlCrossRefPubMedWeb of Science
  37. 37.↵
    L. Ma, S. Wang, D. Wang, M. R. Parsek, D. J. Wozniak, The roles of biofilm matrix polysaccharide Psl in mucoid Pseudomonas aeruginosa biofilms. FEMS immunology and medical microbiology 65, 377–380 (2012); published online EpubJul (10.1111/j. 1574-695X.2012.00934.x).
    OpenUrlPubMed
  38. 38.↵
    L. Ma, M. Conover, H. Lu, M. R. Parsek, K. Bayles, D. J. Wozniak, Assembly and development of the Pseudomonas aeruginosa biofilm matrix. PLoSPathog 5, e1000354 (2009); published online EpubMar (10.1371/journal.ppat.1000354).
    OpenUrl
  39. 39.↵
    W. Khan, S. P. Bernier, S. L. Kuchma, J. H. Hammond, F. Hasan, G. A. O’Toole, Aminoglycoside resistance of Pseudomonas aeruginosa biofilms modulated by extracellular polysaccharide. International microbiology: the official journal of the Spanish Society for Microbiology 13, 207–212 (2010); published online EpubDec (
    OpenUrl
  40. 40.↵
    K. M. Colvin, Y. Irie, C. S. Tart, R. Urbano, J. C. Whitney, C. Ryder, P. L. Howell, D. J. Wozniak, M. R. Parsek, The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofilm matrix. Environ Microbiol 14, 1913–1928 (2012); published online EpubAug (10.1111/j.1462–2920.2011.02657.x).
    OpenUrlCrossRefPubMedWeb of Science
  41. 41.↵
    M. S. Byrd, B. Pang, W. Hong, E. A. Waligora, R. A. Juneau, C. E. Armbruster, K. E. Weimer, K. Murrah, E. E. Mann, H. Lu, A. Sprinkle, M. R. Parsek, N. D. Kock, D. J. Wozniak, W. E. Swords, Direct evaluation of Pseudomonas aeruginosa biofilm mediators in a chronic infection model. Infection and immunity 79, 3087–3095 (2011); published online EpubAug (10.1128/IAI.00057–11).
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    O. Mazur, J. Zimmer, Apo- and cellopentaose-bound structures of the bacterial cellulose synthase subunit BcsZ. J Biol Chem 286, 17601–17606 (2011); published online EpubMay 20 (10.1074/jbc.M111.227660).
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    A. J. Spiers, J. Bohannon, S. M. Gehrig, P. B. Rainey, Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Molecular microbiology 50, 15–27 (2003); published online EpubOct (
    OpenUrlCrossRefPubMedWeb of Science
  44. 44.
    N. L. Schiller, S. R. Monday, C. M. Boyd, N. T. Keen, D. E. Ohman, Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli. J Bacteriol 175, 4780–4789 (1993); published online EpubAug (
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    V. K. Koseoglu, C. Heiss, P. Azadi, E. Topchiy, Z. T. Guvener, T. E. Lehmann, K. W. Miller, M. Gomelsky, Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c-di-GMP-dependent regulation. Molecular microbiology 96, 728–743 (2015); published online EpubMay (10.1111/mmi. 12966).
    OpenUrlCrossRefPubMed
  46. 46.↵
    X. Wang, J. F. Preston, 3rd, T. Romeo, The pgaABCD locus of Escherichia coli promotes the synthesis of a polysaccharide adhesin required for biofilm formation. J Bacteriol 186, 2724–2734 (2004); published online EpubMay
    OpenUrlAbstract/FREE Full Text
  47. 47.↵
    J. B. Kaplan, C. Ragunath, N. Ramasubbu, D. H. Fine, Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity. Journal of bacteriology 185, 4693–4698 (2003); published online EpubAug (
    OpenUrlAbstract/FREE Full Text
  48. 48.↵
    R. Nijland, M. J. Hall, J. G. Burgess, Dispersal of biofilms by secreted, matrix degrading, bacterial DNase. PloS one 5, e15668 (2010)10.1371/journal.pone.0015668).
    OpenUrlCrossRefPubMed
  49. 49.↵
    P. Baker, G. B. Whitfield, P. J. Hill, D. J. Little, M. J. Pestrak, H. Robinson, D. J. Wozniak, P. L. Howell, Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals that its Levels are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation. The Journal of biological chemistry, (2015); published online EpubSep 30 (10.1074/jbc.M115.674929).
  50. 50.↵
    B. H. Park, T. V. Karpinets, M. H. Syed, M. R. Leuze, E. C. Uberbacher, CAZymes Analysis Toolkit (CAT): web service for searching and analyzing carbohydrate-active enzymes in a newly sequenced organism using CAZy database. Glycobiology 20, 1574–1584 (2010); published online EpubDec (10.1093/glycob/cwq106).
    OpenUrlCrossRefPubMedWeb of Science
  51. 51.↵
    J. Tam, G. L. Beilhartz, A. Auger, P. Gupta, A. G. Therien, R. A. Melnyk, Small molecule inhibitors of Clostridium difficile toxin B-induced cellular damage. Chemistry & biology 22, 175–185 (2015); published online EpubFeb 19 (10.1016/j.chembiol.2014.12.010).
    OpenUrlCrossRefPubMed
  52. 52.↵
    S. Roveta, A. Marchese, G. C. Schito, Activity of daptomycin on biofilms produced on a plastic support by Staphylococcus spp. International journal of antimicrobial agents 31, 321328 (2008); published online EpubApr (10.1016/j.ijantimicag.2007.11.012).
    OpenUrl
  53. 53.
    M. E. Zegans, D. Wozniak, E. Griffin, C. M. Toutain-Kidd, J. H. Hammond, A. Garfoot, J. S. Lam, Pseudomonas aeruginosa exopolysaccharide Psl promotes resistance to the biofilm inhibitor polysorbate 80. Antimicrobial agents and chemotherapy 56, 4112–4122 (2012); published online EpubAug (10.1128/AAC.00373–12).
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, J. S. Webb, Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. Journal of bacteriology 188, 7344. 7353 (2006); published online EpubNov (10.1128/JB.00779–06).
  55. 55.↵
    D. G. Davies, C. N. Marques, A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. Journal of bacteriology 191, 1393–1403 (2009); published online EpubMar (10.1128/JB.01214–08).
    OpenUrlAbstract/FREE Full Text
  56. 56.↵
    N. Barraud, D. Schleheck, J. Klebensberger, J. S. Webb, D. J. Hassett, S. A. Rice, S. Kjelleberg, Nitric oxide signaling in Pseudomonas aeruginosa biofilms mediates phosphodiesterase activity, decreased cyclic di-GMP levels, and enhanced dispersal. Journal of bacteriology 191, 7333–7342 (2009); published online EpubDec (10.1128/JB.00975–09).
    OpenUrlAbstract/FREE Full Text
  57. 57.↵
    A. Rahmani-Badi, S. Sepehr, H. Fallahi, S. Heidari-Keshel, Dissection of the cis-2-decenoic acid signaling network in Pseudomonas aeruginosa using microarray technique. Frontiers in microbiology 6, 383 (2015)10.3389/fmicb.2015.00383).
    OpenUrl
  58. 58.↵
    J. B. Kaplan, Therapeutic potential of biofilm-dispersing enzymes. The International journal of artificial organs 32, 545–554 (2009); published online EpubSep (
    OpenUrlPubMed
  59. 59.
    A. Gokcen, A. Vilcinskas, J. Wiesner, Biofilm-degrading enzymes from Lysobacter gummosus. Virulence 5, 378–387 (2014); published online EpubApr 1 (10.4161/viru.27919).
    OpenUrl
  60. 60.↵
    J. B. Kaplan, Biofilm matrix-degrading enzymes. Methods in molecular biology 1147, 203213 (2014)10.1007/978–1–4939–0467–9_14).
    OpenUrl
  61. 61.↵
    J. B. Kaplan, C. Ragunath, K. Velliyagounder, D. H. Fine, N. Ramasubbu, Enzymatic detachment of Staphylococcus epidermidis biofilms. Antimicrobial agents and chemotherapy 48, 2633–2636 (2004); published online EpubJul (10.1128/AAC.48.7.2633–2636.2004).
    OpenUrlAbstract/FREE Full Text
  62. 62.
    J. H. Park, J. H. Lee, M. H. Cho, M. Herzberg, J. Lee, Acceleration of protease effect on Staphylococcus aureus biofilm dispersal. FEMS microbiology letters 335, 31–38 (2012); published online EpubOct (10.1111/j.1574–6968.2012.02635.x).
    OpenUrlCrossRefPubMedWeb of Science
  63. 63.
    T. K. Lu, J. J. Collins, Dispersing biofilms with engineered enzymatic bacteriophage. Proceedings of the National Academy of Sciences of the United States of America 104, 11197–11202 (2007); published online EpubJul 3 (10.1073/pnas.0704624104).
    OpenUrlAbstract/FREE Full Text
  64. 64.↵
    A. S. Messiaen, H. Nelis, T. Coenye, Investigating the role of matrix components in protection of Burkholderia cepacia complex biofilms against tobramycin. Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society 13, 56–62 (2014); published online EpubJan (10.1016/j.jcf.2013.07.004).
    OpenUrl
  65. 65.↵
    G. V. Tetz, N. K. Artemenko, V. V. Tetz, Effect of DNase and antibiotics on biofilm characteristics. Antimicrobial agents and chemotherapy 53, 1204–1209 (2009); published online EpubMar (10.1128/AAC.00471–08).
    OpenUrlAbstract/FREE Full Text
  66. 66.↵
    S. R. Hymes, T. M. Randis, T. Y. Sun, A. J. Ratner, DNase inhibits Gardnerella vaginalis biofilms in vitro and in vivo. The Journal of infectious diseases 207, 1491–1497 (2013); published online EpubMay 15 (10.1093/infdis/jit047).
    OpenUrlCrossRefPubMed
  67. 67.↵
    C. B. Whitchurch, T. Tolker-Nielsen, P. C. Ragas, J. S. Mattick, Extracellular DNA required for bacterial biofilm formation. Science 295, 1487 (2002); published online EpubFeb 22 (10.1126/science.295.5559.1487).
    OpenUrlFREE Full Text
  68. 68.↵
    Q. M. Parks, R. L. Young, K. R. Poch, K. C. Malcolm, M. L. Vasil, J. A. Nick, Neutrophil enhancement of Pseudomonas aeruginosa biofilm development: human F-actin and DNA as targets for therapy. Journal of medical microbiology 58, 492–502 (2009); published online EpubApr (10.1099/jmm.0.005728–0).
    OpenUrlCrossRefPubMedWeb of Science
  69. 69.↵
    B. R. Borlee, A. D. Goldman, K. Murakami, R. Samudrala, D. J. Wozniak, M. R. Parsek, Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix. Molecular microbiology 75, 827–842 (2010); published online EpubFeb (10.1111/j.1365–2958.2009.06991.x).
    OpenUrlCrossRefPubMed
  70. 70.↵
    S. Wang, X. Liu, H. Liu, L. Zhang, Y. Guo, S. Yu, D. J. Wozniak, L. Z. Ma, The exopolysaccharide Psl-eDNA interaction enables the formation of a biofilm skeleton in Pseudomonas aeruginosa. Environmental microbiology reports 7, 330–340 (2015); published online EpubApr (10.1111/1758–2229.12252).
    OpenUrl
  71. 71.↵
    S. C. Chew, B. Kundukad, T. Seviour, J. R. van der Maarel, L. Yang, S. A. Rice, P. Doyle, S. Kjelleberg, Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides. mBio 5, e01536–01514 (2014)10.1128/mBio.01536–14).
    OpenUrlPubMed
  72. 72.↵
    S. Periasamy, H. A. Nair, K. W. Lee, J. Ong, J. Q. Goh, S. Kjelleberg, S. A. Rice, Pseudomonas aeruginosa PAO1 exopolysaccharides are important for mixed species biofilm community development and stress tolerance. Frontiers in microbiology 6, 851 (2015)10.3389/fmicb.2015.00851).
    OpenUrl
  73. 73.↵
    C. K. Stover, X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, M. V. Olson, Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964 (2000); published online EpubAug 31 (10.1038/35023079).
    OpenUrlCrossRefPubMedWeb of Science
  74. 74.↵
    P. G. Bagos, E. P. Nikolaou, T. D. Liakopoulos, K. D. Tsirigos, Combined prediction of Tat and Sec signal peptides with hidden Markov models. Bioinformatics 26, 2811–2817 (2010); published online EpubNov 15 (10.1093/bioinformatics/btq530).
    OpenUrlCrossRefPubMedWeb of Science

Supplementary References

  1. ↵
    Ma, L., Lu, H., Sprinkle, A., Parsek, M. R. & Wozniak, D. J. Pseudomonas aeruginosa Psl is a galactose- and mannose-rich exopolysaccharide. Journal of bacteriology 189, 8353–8356, doi: 10.1128/JB.00620-07 (2007).
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Jennings, L. K. et al. Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix. Proceedings of the National Academy of Sciences of the United States of America 112, 11353–11358, doi:10.1073/pnas.1503058112 (2015).
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Merritt, J. H., Kadouri, D. E. & O’Toole, G. A. Growing and analyzing static biofilms. Current protocols in microbiology Chapter 1, Unit 1B 1, doi: 10.1002/9780471729259.mc01b01s00 (2005).
    OpenUrlCrossRef
  4. Holloway, B. W. Genetic recombination in Pseudomonas aeruginosa. Journal of general microbiology 13, 572–581 (1955).
    OpenUrlCrossRefPubMedWeb of Science
  5. Ma, L., Jackson, K. D., Landry, R. M., Parsek, M. R. & Wozniak, D. J. Analysis of Pseudomonas aeruginosa conditional psl variants reveals roles for the psl polysaccharide in adhesion and maintaining biofilm structure postattachment. Journal of bacteriology 188, 8213–8221, doi:10.1128/JB.01202-06 (2006).
    OpenUrlAbstract/FREE Full Text
  6. Baker, P. et al. Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals that its Levels are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation. The Journal of biological chemistry, doi:10.1074/jbc.M115.674929 (2015).
    OpenUrlAbstract/FREE Full Text
  7. Colvin, K. M. et al. PelA deacetylase activity is required for Pel polysaccharide synthesis in Pseudomonas aeruginosa. Journal of bacteriology 195, 2329–2339, doi:10.1128/JB.02150-12 (2013).
    OpenUrlAbstract/FREE Full Text
  8. Rahme, L. G. et al. Common virulence factors for bacterial pathogenicity in plants and animals. Science 268, 1899–1902 (1995).
    OpenUrlAbstract/FREE Full Text
  9. Wolfgang, M. C. et al. Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences of the United States of America 100, 8484–8489, doi: 10.1073/pnas.0832438100 (2003).
    OpenUrlAbstract/FREE Full Text
Back to top
Posted November 26, 2015.
Download PDF
Email
Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms
Perrin Baker, Preston J. Hill, Brendan D. Snarr, Noor Alnabelseya, Mathew J. Pestrak, Mark J. Lee, Laura K. Jennings, John Tam, Roman Melnyk, Matthew R. Parsek, Donald C. Sheppard, Daniel J. Wozniak, P. Lynne Howell
bioRxiv 032714; doi: https://doi.org/10.1101/032714
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area