Magnetic Immobilization of Dispersin B with Activity in Degradation of Bacterial Biolm

Dispersin B (DspB) is a member of glycoside hydrolase family 20 (GH20) and catalyzes degradation of biolms forming by pathogenic bacteria such as Staphylococcus aureus. Magnetoreceptor (MagR) is a magnetic protein that can be used as a fusion partner for functionally immobilizing proteins on magnetic surfaces. In the present study, a recombinant protein DspB-MagR was constructed by fusing MagR to the C-terminus of DspB and expressed in Escherichia coli. Magnetic immobilization of puried DspB-MagR on Fe 3 O 4 @SiO 2 nanoparticles was achieved and characterized by means of various techniques including SDS-PAGE, FTIR spectrometry, TGA measurements, zeta potential measurement, and SEM analysis. Stability and activity of immobilized DspB-MagR on Fe 3 O 4 @SiO 2 nanoparticles were analyzed under different conditions such as temperature, pH, and storage time. Removal of biolms forming by Staphylococcus aureus and other medical source bacterial species were achieved by using Fe 3 O 4 @SiO 2 nanoparticles loading with DspB-MagR. This work promoted potential applications of DspB and similar enzymes for medical purposes.


Introduction
Bio lms are a group of bacteria surrounding by extracellular polymeric matrix (EPS) composed of polysaccharides, lipids and nucleic acids [1]. Bacteria take advantages of bio lms in reducing sensibility to antibiotics. Antibiotic resistance of bacteria in bio lm can be 100 to 1000 folds higher than that of planktonic cells [2]. Bio lm formation on medical devices especially implanting devices is a critical problem in healthcare and has inevitable consequences of serious infections and failure of therapy [3].
Bacterial bio lms were involved in over 65% of bacterial infections [4], which are normally caused by Streptococcus species, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus, Enterococcus and Candida spp. [4]. Although different bacteria can form different types of bio lms, poly-beta(1,6)-N-acetylglucosamine (PNAG) is the major polysaccharide component of EPS to most of pathogenic bacteria [5] such as Staphylococcus epidermidis, Staphylococcus aureus and A. actinomycetemcomitans [6]. Therefore, techniques targeting to PNAG are useful in detaching bacterial bio lms.
Bacterial cells within mature bio lm can be released into external environment and attach on another surface to form new bio lm. The beta-N-acetylglucosamine enzyme Dispersin B (DspB) plays important roles during this process [7]. DspB is a hydrolase of PNAG and has functions in bio lm degradation that promotes resistance to antibiotics such as teicoplanin, rifampicin, clindamycin, gentamicin and cipro oxacin [8,9]. Scientists attempted to immobilize DspB on biocompatible surface and carriers, which improved antibio lm activity of DspB [10][11][12][13]. However, these approaches mainly rely on chemical modi cations of carries and chemical conjugations between enzymes and carriers, which may raise safety issues for medical applications.
Magnetoreceptor (MagR) is a magnetic protein that can interact with external magnetic elds [14]. MagR was explored as a fusion tag with enzymes for functional immobilization of these enzymes on Fe 3 O 4 and Fe 3 O 4 @SiO 2 nanoparticles [15,16]. Magnetic interactions between MagR and supports reduced in uences of chemical environment and facilitated activities of enzymes over a broad range of environmental conditions. Therefore, it provides an alternative physical means for immobilizing DspB that has less chemical toxicity.
The aim of this study is to take advantages of MagR as the fusion partner of DspB to facilitate functional immobilization of DspB on magnetic Fe 3 O 4 @SiO 2 particles, which can be used for inhibiting and removing bio lms. Activity of recombinant DspB-MagR in degrading bacterial bio lms was evaluated before and after immobilization. Expression and puri cation of DspB-MagR was based on the pET System Manual (Novagen, German).
Brie y, a single colony of Escherichia coli BL21(DE3) harbouring pET28a[DspB-MagR] was inoculated into Luria-Bertani (LB) medium containing 0.05 mg ml − 1 kanamycin and shaking at 37 o C overnight. The cell suspension was inoculated into LB medium and followed by shaking at 37 o C until an optical density at 600 nm (OD600) of 0.5-0.6 was reached. Protein expression was initiated by supplying with 1 mM isopropyl-p-D-thiogalactoside (IPTG) and continuing shaking at 16 o C for 24 hours. Cells were harvested by centrifugation at 6,000 g, 4 o C for 10 minutes, re-suspended in lysis buffer (50 mM NaH 2 PO 4 , 10 mM imidozale, pH 8.0/300 mM NaCl), and lysed by using a high-pressure cell disruptor TSO.75 KW (Constant Systems Ltd., UK). The cell lysate was centrifuged at 12,000 g for 10 minutes to remove cell debris and subjected to Ni-NTA a nity chromatography. The Ni-NTA a nity resin was pre-washed and equilibrated with wash buffer (50 mM NaH 2 PO 4 , 20 mM imidozale, 300 mM NaCl, pH 8). Unbound proteins were washed three times with wash buffer. Bound DspB-MagR was eluted with elution buffer (50 mM NaH 2 PO 4 , 300 mM imidozale, 300 mM NaCl, pH 8.0). were collected by using a magnet and washed three times with ethanol and deionized water, and then dried under vacuum overnight.

Magnetic Immobilization of DspB-MagR
To immobilize DspB-MagR on Fe 3 O 4 @SiO 2 nanoparticles, 1 ml solution containing DspB-MagR was mixed with 10 mg nanoparticles for 30 minutes. The nanoparticles with loaded Dsp-MagR was collected by centrifugation at 3000 g for 60 seconds and washed twice with 1 ml PBS (pH 6.0) to remove unloaded enzyme.
Enzymatic Activity Assay of DspB-MagR Enzymatic activity assay of DspB-MagR was based on Tan et al. (2015) [12] by using 4-nitrophenyl-Nacetyl-beta-D-glucosaminide as the substrate to produce chromogenic pnitrophenolate. Brie y, 10 µl solution containing DspB-MagR were mixed with 0.2 ml substrate solution (50 mM sodium phosphate buffer containing 100 mM NaCl, 5 mM substrate, pH 6.0) and incubated at 37 o C for 10 minutes. The reaction was terminated by adding with 5 µl 10M NaOH. The produced p-nitrophenolate was monitored by measuring the increase of optical density.
Sensitivity of DspB-MagR to pH was investigated by assaying the enzymatic activity in disodium hydrogen phosphate-citric acid buffer (pH 3-8) and glycine sodium hydroxide buffer (pH 9-10).
Sensitivity of DspB-MagR to temperature was investigated by assaying the enzymatic activity in sodium phosphate buffer (pH 6.0) at temperature range from 4 o C to 45 o C. Stability of DspB-MagR over time was evaluated by comparing the initial activity with the residual activity after incubation for various periods.

Assaying the Formation and Degradation of Bio lm
Bacterial species, that is, one standard stain of Staphylococcus aureus and three bacterial species from replaced knee implants of patients with infections (Union Hospital, Wuhan, China), as well as bacterial mixtures from medical practice were explored for bio lm formation and degradation. Isolation of bacteria from medical practice were based on the majority and morphology of colonies on streaking plates. The isolated bacteria were characterized by 16s RNA sequencing, which showed that these stains are Staphylococcus sp., Bacillus Cereus, and Pseudomonas Putida.
For growing biobilms, bacteria were cultured overnight and diluted to 0.05 OD600 with LB medium.
Aliquots (2 ml) were transferred to a 24-well plate. The plate was incubated at 37 o C for 24 hours and washed three times with LB medium to remove planktonic cells.
For assaying the formation of bio lms, bio lms were stained with 1 ml of 0.1% (w/v) crystal violet solution for 10 to 20 minutes, and washed three times with PBS (pH 7.0). The crystal violet remained by the bio lm was dissolved by adding 200 µl 30% (v/v) acetic acid and diluted to 1 ml before measuring optical density at 590 nm.
For assaying bio lm degradation, the formed bio lms were treated with DspB-MagR loaded Fe 3 O 4 @SiO 2 (10 mg/ml) and/or lysozyme (1 mg/ml) for an hour at 37 o C. Then the residual bio lms were assayed as described above. Triplicated measurements were performed for each assay.

Construction and Expression of DspB-MagR
The magR gene were ampli ed using a pair of primers containing restriction site of endonuclease XhoI and then inserted at the downstream of dspB gene to construct the plasmid pET(DspB-MagR) (Fig. 1, Panel A), with a sequence encoding hexahistidine tag at the C-terminus of the recombinant protein DspB-MagR to facilitate the a nity puri cation.
Expression of DspB-MagR was induced by supplying IPTG to the culture containing E. coli host BL21 harboring the vector pET(DspB-MagR). Bacterial growth was signi cantly reduced upon the addition of IPTG (Fig. 1, Panel B), implying the involvement of bacterial metabolism in synthesizing the heterogeneric DspB-MagR retarded the bacterial growth. Expression of recombinant DspB-MagR was con rmed with SDS-PAGE electrophoresis (Fig. 1, Panel C), which showed that there was overexpression of protein with an apparent molecular weight between 50 and 70 kDa. This protein was assigned to be DspB-MagR with a theoretic molecular weight of 60 kDa.
Fast overexpression of heterogeneric protein sometimes leads to formation of inclusion body containing proteins of interests that are not correctly folded. In the present study, IPTG-induced DspB-MagR expression was attempted at two temperatures (that is, 16 o C and 37 o C) to avoid the formation of DspB-MagR inclusion body. It was observed that DspB-MagR expression level at 16 o C was lower than that at 37 o C. However, the soluble version of DspB-MagR at 16 o C was signi cantly improved (Fig. 1, Panel D). It might be due to that bacterial protein synthesis is slow at low temperature, which provides translated DspB-MagR with more time to fold properly.

Puri cation and Immobilization of DspB-MagR
Expressed DspB-MagR was puri ed by using Ni-NTA a nity chromatography and immobilized on Fe 3 O 4 @SiO 2 nanoparticles (Fig. 2, Panel A). The homogeneity of DspB-MagR was over 95% after puri cation and immobilization as determined by densitometric scanning of Coomassie blue stained SDS-polyacrylamide gels. SEM revealed no signi cant morphological changes of Fe 3 O 4 @SiO 2 nanoparticles before and after loading with DspB-MagR ( Figure S1, see Supporting Information). FTIR spectrometry of Fe 3 O 4 @SiO 2 nanoparticles with or without DspB-MagR loading showed that there were characteristic bands of Si-O-Si band at 1095 cm − 1 and Fe-O band at 596 cm − 1 , 576 cm − 1 (Fig. 2, Panel  B). There was a shift of absorbance peak from 1634 cm − 1 (before protein loading) to 1652 cm − 1 (after protein loading), which was attributed to in uence of the amine of polypeptide and proved the immobilization of DspB-MagR on Fe 3 O 4 @SiO2 nanoparticles. Immobilization of DspB-MagR on Fe 3 O 4 @SiO 2 nanoparticles was also veri ed by TGA measurement (Fig. 2, Panel C). There was a weight loss of 3% and 5% for both Fe 3 O 4 @SiO 2 nanoparticles with or without DspB-MagR loading when raising temperature up to 150 o C, which was due to water evaporation. When raising temperature from 150 o C to 350 o C, a signi cant weight loss of 1.25% was observed for Fe 3 O 4 @SiO 2 nanoparticles loading with DspB-MagR due to the thermal decomposition of protein. Therefore, the loading capacity of Fe 3 O 4 @SiO 2 nanoparticles to DspB-MagR was estimated as ~ 1.25 mg DspB-MagR per 100 mg nanoparticles. Zeta potential of Fe 3 O 4 @SiO 2 nanoparticles with or without DspB-MagR loading was measured at different pH ( Fig. 2, Panel D). Zeta potential is the electrostatic potential that exists at the shear plane of a particle, which is related to both surface charge and the particle's local environment. It was observed that after loading with DspB-MagR, Fe 3 O 4 @SiO 2 nanoparticles exhibited higher zeta potential than that of naked Fe 3 O 4 @SiO 2 nanoparticles. This could be attributed to the ionization of functional NH 2 groups of DspB-MagR protein.

Bioactivity of Immobilized DspB-MagR
Enzymatic activity of DspB-MagR before and after loading on Fe 3 O 4 @SiO 2 nanoparticles was investigated under various conditions. Generally, the activity of enzymes is affected by factors such as pH, temperature, functional time [18]. It was observed that DspB-MagR exhibited similar spectrum of sensitivity to pH before and after loading on Fe 3 O 4 @SiO 2 nanoparticles (Fig. 3, Panel A). Both showed highest activity in pH 6. Therefore, immobilization on Fe 3 O 4 @SiO 2 nanoparticles did not change the pH sensitivity of DspB-MagR.
Sensitivity of DspB-MagR to temperature changed after loading on Fe 3 O 4 @SiO 2 (Fig. 3, Panel B). Before immobilization, DspB-MagR exhibited highest activity at 30 o C. While the immobilized DspB-MagR exhibited highest activity at 37 o C that is the regular body temperature of human, implying that immobilization of DspB-MagR on Fe 3 O 4 @SiO 2 nanoparticles is suited to medical purposes.
Stability of DspB-MagR over time was investigated before and after loading on Fe 3 O 4 @SiO 2 nanoparticles (Fig. 3, Panel C). There were two phases of activity changes for both enzymes over time after incubation at their optimal temperature (30 o C for unloaded DspB-MagR and 37 o C for immobilized DspB-MagR). In the rst two to three hours of incubation, both enzymes suffered a quick decrease and increase of the activity. Although the activity of both enzymes kept decreasing after three hours of incubation, immobilized DspB-MagR showed higher activity of than that of unloaded ones at sampling time, implying that immobilization of DspB-MagR on Fe 3 O 4 @SiO 2 nanoparticles is bene cial for retaining the activity of DspB-MagR over time.

Degradation of Bio lm by Immobilized DspB-MagR
Bioactivity of DspB-MagR before and after loading on Fe 3 O 4 @SiO 2 was assayed for detaching bacterial bio lms (Fig. 4). When testing with the standard strain of Staphylococcus aureus (SA), naked Fe 3 O 4 @SiO 2 nanoparticles did not exhibit any effects on removing bio lm. Unloaded DspB-MagR showed limited function and removed about 10% of bio lm. After loading on Fe 3 O 4 @SiO 2 nanoparticles, activity of DspB-MagR on removing bio lm was signi cantly improved and detached more than 50% of bio lm.
Activity of DspB-MagR loading on Fe 3 O 4 @SiO 2 nanoparticles was further examined on removing bio lms forming by bacteria samples from clinic source, that is, one mixed bacterial species (MB) and three isolated and characterized species, Staphylococcus sp. (SS), Bacillus Cereus (BC), and Pseudomonas Putida (PP). Although it was not signi cant, immobilized DspB-MagR showed activity in degrading bio lm forming by mixed bacterial species or Pseudomonas Putida. As to Staphylococcus sp. or Bacillus Cereus, immobilized DspB-MagR exhibited signi cant activity on detaching bio lm, over 40% (Bacillus Cereus) or 60% (Staphylococcus sp.) of bio lms were removed.

Conclusion
In this study, a recombinant protein DspB-MagR was constructed by fusing a magnetic protein MagR to the C-terminus of DspB. Functional expression and puri cation of the resultant DspB-MagR was achieved. The magnetic protein MagR was served as a magnetic partner to facilitate immobilization of DspB-MagR on magnetic nanoparticles. The immobilization enhanced the storage stability of DspB-MagR. The optimum temperature of DspB-MagR was shifted from 30 o C (DspB alone) to 37 o C (DspB-MagR), which provides advantages of DspB in medical practice. The magnetically immobilized DspB-MagR showed signi cant activity to detach bio lms forming by either standard bacterial strain of Staphylococcus aureus or bacterial species Staphylococcus sp. and Bacillus Cereus from clinic practice. Although it is not signi cant, the immobilized DspB-MagR showed activity to remove bio lms forming by mixed bacterial species or Pseudomonas Putida from clinic practice. These results suggest that DspB-MagR immobilizing on magnetic nanoparticles can be used for preventing and treating medical deviceassociated infections. Moreover, the complex of DspB-MagR and magnetic nanoparticles can be controlled and distributed under the function of magnetic force, which will further broaden its range of applications.

Declarations
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