Peptidoglycan binding protein (PGBP)-modified magnetic nanobeads for efficient magnetic capturing of Staphylococcus aureus associated with sepsis in blood

Peptidoglycan-binding protein-modified magnetic nanobeads (PGBP-MNBs) were prepared for efficient magnetic capturing of Staphylococcus aureus (S. aureus), which is associated with sepsis, using the binding affinity of PGBP for the peptidoglycan (PG) layer on S. aureus. These PGBP-MNBs can simply capture S. aureus in plasma within 1 hr or even 15 min. Importantly, they also can capture various types of Gram-positive bacteria, such as Bacillus cereus and methicillin-resistant and methicillin-susceptible S. aureus (MRSA and MSSA). We believe that PGBP-based systems will be used to develop diagnostic systems for Gram-positive bacteria-related diseases.

approximately 421 amino acid. We also tagged PGBP with green fluorescent protein (GFP) to impart green fluorescence (Fig. 2a). The affinity of PGBP for the PG layer of Gram-positive S. aureus bacteria, which was measured as the binding affinity (K D ), was determined using a BLItz system. The result showed that the K D was 6.49 nM (Fig. S2). In addition, we visually confirmed the specific binding affinity of PGBP for PG layer from S. aureus by fluorescence microscopy analysis. S. aureus bacteria stained with red fluorescent reagents were incubated with PGBP (green fluorescence). After 1 hr, the bacteria were purified by centrifugation to eliminate unbound PGBP and then were re-suspended in buffer containing 10% plasma, similar to physiological blood conditions in humans (Fig. 2b). As shown in Fig. 2c, the red and green fluorescence signals were colocalized, indicating that PGBP was bound to S. aureus. It was confirmed in fluorescence spectra as well that PGBP/S. aureus showed both green and red fluorescence intensities (Fig. S3). PGBP-MNBs were prepared for magnetic capturing of S. aureus at room temperature. The bacteria were then enriched by applying a magnet, as Ni-NTA was immobilized on the MNBs along with PGBP [43][44][45][46] . Histidine (His)-tags on PGBP act as a chelating agent and form chelate complexes with nickel (Ni) ions from Ni-NTA, which offer vacant electron orbitals to form coordinate bonds. The size of the MNBs before PGBP binding was approximately 900 nm, and their size after PGBP binding increased to approximately 1 μm (Fig. 3a). After reaction of the PGBP-MNBs with S. aureus in buffer containing 10% plasma for 1 hr, the resulting PGBP-MNBs/S. aureus complex was dropped on a glass slide, and then a magnet was placed under this glass slide. The complex was magnetically manipulated by the external magnetic field, showing a yellow fluorescence signal overlapped with both the red and green fluorescence signals (Fig. 3b,c). In addition, we recorded fluorescence spectra of the PGBP-MNBs/S. aureus complex at excitation wavelengths of 488 and 588 nm using a multimode-microplate reader to confirm the binding capacity of PGBP-MNB for S. aureus. Free PGBP, MNBs and S. aureus were also measured in the same manner as controls (Fig. S3). The PGBP-MNB/S. aureus complex showed fluorescence intensities corresponding GFP, which is similar to free PGBP (Figs 3d and S3). In addition, at the excitation wavelength of 588 nm, PGBP-MNBs/S. aureus exhibited fluorescence signals corresponding to S. aureus. As expected, as the reaction time increased, the fluorescence intensity under 588 nm excitation increased, indicating that the amount of S. aureus captured by the PGBP-MNBs was increased (Figs 3e and S3). Therefore, the ability of PGBP-MNBs to capture S. aureus could increase with the reaction time. Furthermore, for quick acquisition of S. aureus by magnetic concentration using these PGBP-MNBs, we evaluated the capturing abilities   for Type Cultures (KCTC), and clinically isolated MRSA (#77, #78, #79 and #80) and MSSA (#85, #86, #87 and #88) were obtained from BioNano Health Guard Research Center (H-GUARD). Bacteria (10 3.7 CFU/mL) were separately mixed with PGBP-MNBs for 1 hr at room temperature. After incubation, the unbound bacteria were removed by magnetically assisted washing, and then the captured bacterial concentrations were measured by real-time PCR assay. The data revealed the great efficacy of the PGBP-MNBs in magnetic capturing of approximately 10 2.8~1 0 4 CFU/mL Gram-positive bacteria with a capture efficiency of about 81.67% 53 . These results confirmed that PGBP-MNBs could be used universally to efficiently detect most Gram-positive bacteria.

Conclusions
We developed PGBP-MNB as a magnetic capturing probe of Gram-positive S. aureus, which is associated with sepsis, and confirmed its ability to capture these bacteria within just 15 min at room temperature. In particular, since there is a PG layer on Gram-positive bacteria, the PGBP-MNBs can capture not only S. aureus but also B. cereus, MRSA and MSSA. Notably, because it uses a protein (PGBP) that binds universally to Gram-positive bacteria, this probe (PGBP-MNBs) has increased practicality compared to probes that increase selectivity by using  an antibody that binds to a specific bacterium. Furthermore, we expect this PGBP-based capture platform to be applicable to diagnostic systems for bacteria-related diseases.

Experimental Section
Chemicals. We Table S1.
Cloning, expression and purification of PGBP. We constructed a green fluorescence protein (GFP)-tagged peptidoglycan binding protein (PGBP) vector to express the fusion protein of PGBP and GFP (Fig. S1).

Binding capacity of PGBP for S. aureus (PGBP/S. aureus).
We measured the binding affinity between PGBP and S. aureus using a biolayer interferometry-based biosensor BLItz system (FORTEBIO). PG from S. aureus was purchased from SIGMA-ALDRICH. PGBP loaded on Ni-NTA biosensors to bind with 6X histidine of PGBP and Ni-NTA, equilibrated in PBS for 1 min to establish a stable baseline, and then dipped into 4uL of PG from S. aureus (0 ~ 50 nM) to obtain the association curve for 300 s. The dissociation curve was obtained for 300 s using dipping holder. Afterwards, Binding affinities were calculated by fitting the curves using BLItz software (Fig. S2). We also performed binding capacity of PGBP against S. aureus in a same manner. Moreover, to visually confirm the binding of PGBP for S. aureus (10 7 CFU/mL), we used a red fluorescent dye (BACLIGHT RED BACTERIAL STAIN) for bacterial staining (Ex: 581-596 nm/Em: 644 nm) (THERMO FISHER SCIENTIFIC). Then, bacteria were co-incubated with PGBP (1 mg/mL, 20 µL) for 1 or 2 hr at 37 °C. After incubation, unbound PGBPs were washed with PBS three times by centrifugation at 10,000 rpm for 10 min. The PGBP/S. aureus complex was observed by Jaewoo Lim using a fluorescent Microscope (EVOS Cell Imaging Systems, THERMO FISHER SCIENTIFIC).

Ability of PGBP-MNBs to capture S. aureus (PGBP-MNBs/S. aureus).
We prepared 10 1.7~1 0 5.7 CFU/ mL S. aureus in human plasma solution (10% human plasma in PBS). Then, 1 mL of PGBP-MNBs (0.1 mg/mL) were injected into the bacterial sample, and then they (PGBP-MNBs/S. aureus) mixed for 15 min, 30 min and 1 hr, respectively. To remove unbound bacteria, the samples were washed three times with PBS using magnetic separation methods. Additionally, we confirmed the capturing ability using various Gram-positive bacteria, including S. aureus, B. cereus, MRSA and MSSA. In these experiments, each type of bacteria (10 3.7 CFU/mL) was separately mixed with the PGBP-MNBs (1 mL, 0.1 mg/mL) for 1 hr, and then unbound bacteria were removed by using magnetic separation methods.

Confirmation of the capture of S. aureus captured by PGBP-MNBs (PGBP-MNBs/S. aureus).
To confirm the capture ability of the PGBP-MNBs, we conducted flow cytometry analysis and real-time PCR. Flow cytometry analysis was performed using a FACSCalibur instrument (BECTON DICKINSON AND CO., USA) and software (WINMD) for data analysis. PGBP was tagged with GFP to impart green fluorescence, and S.
aureus was stained with a red fluorescence dye. In addition, we carried out real-time PCR using a CFX96 Touch ™ DNA and control (16s rRNA) primer sequences were described in previous papers. PCR conditions were in accordance with the product manual of QuantiTect SYBR Green PCR kits (QIAGEN, Germany). We also measured the concentrations of the other types of captured bacteria (B. cereus, MRSA and MSSA) by real-time PCR in the same manner.