A Genetically Engineered Biofilm Material for SARS-CoV-2 Capturing and Isolation.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuously infecting people all around the world since its outbreak in 2019. Studies for numerous infection detection strategies are continuing. The sensitivity of detection methods is crucial to separate people with mild infections from people who are asymptomatic. In this sense, a strategy that would help to capture and isolate the SARS-CoV-2 virus prior to tests can be effective and beneficial. To this extent, genetically engineered biomaterials grounding from the biofilm protein of Escherichia coli are beneficial due to their robustness and adaptability to various application areas. Through functionalizing the E. coli biofilm protein, diverse properties can be attained such as enzyme display, nanoparticle production, and medical implant structures. Here, E. coli species are employed to express major curli protein CsgA and Griffithsin (GRFT) as fusion proteins, through a complex formation using SpyTag and SpyCatcher domains. In this study, a complex system with a CsgA scaffold harboring the affinity of GRFT against Spike protein to capture and isolate SARS-CoV-2 virus is successfully developed. It is shown that the hybrid recombinant protein can dramatically increase the sensitivity of currently available lateral flow assays for Sars-CoV-2 diagnostics.

Introduction

The world has been floundering in the COVID‐19 pandemic caused due to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) since 2019. Starting from the first day, SARS‐CoV‐2 has caused almost 447 million confirmed cases and 6 million deaths.[ ]

The need to detect the SARS‐CoV‐2 infections has never lost its power and continues to increase even more. The infection detection is achieved through real‐time reverse transcription polymerase chain reaction (RT‐PCR), denoted as the golden standard.[ ] In places where people experience resource and qualified staff limitations, other options for SARS‐CoV‐2 infection detection have emerged, and several methods have been reported to work including Lateral Flow Assays (LFA).[ , , ] Although the LFA tests for SARS‐CoV‐2 result faster and require fewer resources in terms of trained personnel and equipment when compared to RT‐PCR, several studies have revealed the lack of sensitivity in LFA tests for detecting SARS‐CoV‐2 infections.[ , , ] Therefore, it is crucial to increase the sensitivity of point‐of‐care rapid tests to be able to detect SARS‐CoV‐2 infections vigorously.

The utilization of naturally found proteins has been a concern regarding their usage as short peptides or proteins. Their abundance and availability in nature have placed them in the center of attention. Their naturally existing varied properties combined with the capability of genetic engineering have supported the emergence of new synthetic proteins with modified characteristics. In this sense, self‐assembling amyloid proteins, which can also be found in fiber form, are concerned as one of the robust proteins due to their high resistance properties in harsh environments.[ ] Their structure composed of hydrogen bonds provided by the cross‐β strands of amyloid proteins provides a strong network within and outstanding material properties, including high Young's modulus and tensile strength.[ , ] Therefore, self‐assembling amyloid proteins are being studied and reported regarding their potential as genetically engineered proteins.

The major curli protein of Escherichia coli, CsgA, is one of the self‐assembling amyloid proteins, which have previously been used as scaffolds due to its robust fiber structure for various applications.[ , ] In our previous studies, we have shown that genetically modified CsgA major curli protein can harbor numerous new properties. We have proven that it can be engineered to provide suitable conditions for prolonged enzyme activity and to have conductive properties, which are considered as the new to nature properties for the curli fiber protein.[ , ] Moreover, engineered curli fibers by fusing them with functional proteins and peptides have been previously used to embed various functions, including fluorescence, virus disinfection, and enzyme display.[ , , ] In fact, these examples prove CsgA as useful for engineered biomaterial structures. The mentioned instances corroborate with the idea that each of these approaches provides unique systems of varying functionalities of CsgA.

In this study, we have developed a system based on the CsgA fiber scaffold. We aimed to interact, capture and precipitate SARS‐CoV‐2 virus particles within a confined volume of a complex pellet via Griffithsin (GRFT) functionalized CsgA proteins. Our approach to functionalizing CsgA protein with GRFT was through a protein–peptide domain. In fact, the protein–peptide domain is a well‐studied duo known as SpyTag and SpyCatcher. The motivation for using SpyTag‐SpyCatcher domains was to irreversibly combine CsgA and GRFT via the isopeptide bond formation occurring between SpyTag and SpyCatcher. We fused SpyTag peptide with CsgA and SpyCatcher protein domain with GRFT to form the two subunits (Figure  ). The subunits were combined together in a convenient reaction medium resulting in the assembly of the complex system, which we named as CsgA‐SpyTag + SpyCatcher‐GRFT (Figure 1b). Latter to trials of the complex system with SARS‐CoV‐2, the system was analyzed in terms of capturing, infectivity, and ability to precipitate the SARS‐CoV‐2 virus particles along with the complex pellet (Figure 1c).

Figure 1

Road map for CsgA‐SpyTag + SpyCatcher‐GRFT complex assembly followed by capture, infectivity analysis, and precipitation analysis latter to incubation with SARS‐CoV‐2. a) SpyCatcher‐GRFT, CsgA‐SpyTag subunits are initially recombinantly expressed. b) CsgA‐SpyTag + SpyCatcher‐GRFT complex assembly is performed once the subunits are obtained. c) The CsgA‐SpyTag + SpyCatcher‐GRFT complex is incubated with SARS‐CoV‐2 virus particles to capture them from aqueous media. Following SARS‐CoV‐2 capture, infectivity and precipitation analysis is done. The figure is created with BioRender.com.

Results and Discussion

CsgA‐SpyTag + SpyCatcher‐GRFT Complex Formation

We investigated the formation of the CsgA‐SpyTag + SpyCatcher‐GRFT complex, starting from its individual subunits and the assembled complex structure at the end (Figure  ). The CsgA‐SpyTag biofilm fibers, were overexpressed in ΔcsgA cells derived from E. coli (MG 1655) using an aTc inducible plasmid. The filtered biofilm fibers were characterized and verified by Transmission Electron Microscopy (TEM) by labeling the samples with Ni‐NTA conjugated Au nanoparticles, which are long known for their affinity against Histidine tags (Figure 2b).[ ] Moreover, we have also confirmed the production of CsgA‐SpyTag biofilm fibers via Congo Red (CR) staining. For β‐sheet rich amyloid structures like CsgA and CsgA‐SpyTag, CR is an amyloid binding dye, which can also be used to quantify amyloid fibers.[ ] Therefore, we used CR staining to confirm the CsgA‐SpyTag fiber formation further and quantify with respect to the cells that are not capable of fiber formation (Figure S1, Supporting Information). The other subunit was SpyCatcher‐GRFT fusion protein, which was overexpressed in E. coli BL21 (DE3) cells via an Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) inducible pET22b (+) plasmid. The SpyCatcher‐GRFT fusion protein was verified using Western Blot analysis, and the corresponding band was observed at 30 kDa, corroboratively (Figure 2c).

Figure 2

Expression of subunits individually and CsgA‐SpyTag + SpyCatcher‐GRFT complex assembly afterward. a) A schematic representation of CsgA‐SpyTag, SpyCatcher‐GRFT subunits and assembled CsgA‐SpyTag + SpyCatcher‐GRFT complex after incubation of subunits in 50 mm Phosphate buffer. The figure is created with BioRender.com. b) Transmission electron microscopy (TEM) image of CsgA‐SpyTag biofilm fibers labeled with Ni‐NTA conjugated Au nanoparticles. The scale bar represents 100 nm. c) Western blot analysis for overexpressed SpyCatcher‐GRFT fusion protein subunit. The SpyCatcher‐GRFT fusion protein was observed at 30 kDa. d) Western blot analysis for CsgA‐SpyTag + SpyCatcher‐GRFT complex. From top to bottom the red arrows represent CsgA‐SpyTag + SpyCatcher‐GRFT complex (45 kDa), SpyCatcher‐GRFT fusion protein (30 kDa) and CsgA‐SpyTag (15 kDa), respectively indicating that the complex assembly was successful.

The SpyTag and SpyCatcher protein domains are shown to form strong intramolecular isopeptide bonds when they are in a favorable reaction media.[ ] Moreover, previous crystallography and Nuclear Magnetic Resonance (NMR) data have proved that the isopeptide bond formation happens between the unprotonated amine group of Lys31 and carbonyl carbon of Asp117, which occurs due to the nucleophilic attack catalyzed by the neighbor Glu77. [ ] Therefore, we aimed to achieve the irreversible isopeptide bond formation between our subunits. Following the production validation for both subunits, we combined them in 50 mm Phosphate Buffer as previously done and verified to form the bond formation.[ ] The resulting complex structure was bound together irreversibly with the isopeptide bond between SpyTag and SpyCatcher protein domains of two subunits.[ ] The structure obtained after combining the subunits was assessed by Western Blot to corroborate the complex formation (Figure 2d). The western blot of the samples taken from the reaction buffer in which the two subunits were gathered to form a complex indicated that the subunits interact and form CsgA‐SpyTag + SpyCatcher‐GRFT complex structure. The result represented CsgA‐SpyTag fibers and SpyCatcher‐GRFT fusion protein separately at 15 and 30 kDa, respectively. Furthermore, CsgA‐SpyTag + SpyCather‐GRFT complex, a 45 kDa product, was observed affirming with the previous studies of a copious number of application areas for bond formation between SpyTag − SpyCatcher domains.[ , , , , ]

SARS‐CoV‐2 Infectivity Assay After Incubation with CsgA‐SpyTag + SpyCatcher‐GRFT Complex

Griffithsin (GRFT) is a protein, i.e., lectin, known for its antiviral properties[ , ] and affinity against mannose groups of surface glycoproteins of enveloped viruses.[ ] The antiviral effect of Griffithsin is known to emerge from the phenomenon of GRFT binding to surface glycoproteins of enveloped viruses and blocking the viral internalization step as a result.[ , ] The antiviral effect of GRFT on several enveloped viruses such as human immunodeficiency virus‐1,[ ] SARS‐CoV,[ ] and middle east respiratory syndrome coronavirus[ ] was shown previously through the interactions of GRFT with their surface proteins gp‐120, and S‐proteins, respectively. In a recent study, GRFT was also shown to be prevalent for binding the spike protein of SARS‐CoV‐2 virus and blocking its entry to Vero6 cell lines and IFNAR−/−mouse models.[ ]

Therefore, following the assembly of the complex system, we investigated the antiviral property of GRFT fused in a system of biofilm fibers, CsgA‐SpyTag + SpyCatcher‐GRFT, via Tissue Culture Infective Dose 50% (TCID50) assay. SARS‐CoV‐2 was incubated with CsgA‐SpyTag + SpyCatcher‐GRFT complex for 30 min, and the supernatant was analyzed following pelleting the fiber complex. The fiber complex pellet was expected to interact and capture SARS‐CoV‐2 virus particles due to the presence of GRFT in the complex (Figure  ), and the supernatant was expected to have less amount of infective SARS‐CoV‐2. Therefore, the supernatant was analyzed by TCID50 assay. Moreover, the assay comprised of four CsgA‐SpyTag + SpyCatcher‐GRFT complexes each formed with different SpyCatcher‐GRFT concentrations (44, 171, 228, 298 µg mL−1). Different concentrations were used to observe the possible differences in capturing of SARS‐CoV‐2 post‐incubation with the complex. With this, we aimed to observe if varying concentrations of SpyCatcher‐GRFT fusion protein will have different effects on capturing efficiency and therefore the total reduction in active virus infectivity by the complex. The complexes were separately incubated with SARS‐CoV‐2 with an initial infective titration of TCID50 = 107.25 mL−1 for 30 min, and the supernatant was used for TCID50 assay. The observed cytopathic effects at the end of the TCID50 assay were used to calculate the total infectivity of remaining SARS‐CoV‐2 post CsgA‐SpyTag + SpyCatcher‐GRFT incubation. The results suggest that the infectivity of SARS‐CoV‐2 was reduced significantly after incubation with CsgA‐SpyTag + SpyCatcher‐GRFT complex compared to the control groups (Figure 3b), indicating that the complex system successfully captures the virus particles and the remaining virus particles in the supernatant (as the captured ones are precipitated with the complex pellet) show a significantly reduced infectivity. On the other hand, SARS‐CoV‐2 infectivity after incubation with complex structures with four different SpyCatcher‐GRFT concentrations did not result in prominent differences compared to each other. Interestingly, we also observed that CsgA‐SpyTag fibers and CsgA‐SpyTag fibers incubated with E. coli BL21 cell lysate are capable of interacting with SARS‐CoV‐2 and interfering with its infectivity to some extent when compared to the initial infective titration of the virus used in the experiments. However, upon comparison with the initial infective titration of the virus (TCID50 = 107.25 mL−1), CsgA‐SpyTag + SpyCatcher‐GRFT complex is significantly effective for capturing the virus, removing from aqueous media and reducing its total infectivity.

Figure 3

Infectivity analysis for SARS‐CoV‐2 after incubation with CsgA‐SpyTag + SpyCatcher‐GRFT complex. a) Schematic representation of captured SARS‐CoV‐2 virus particles with CsgA‐SpyTag + SpyCatcher‐GRFT complex. The figure is created with BioRender.com. b) Infectivity analysis conducted for SARS‐CoV‐2 after incubation with the complex structure. TCID50 analysis was conducted with CsgA‐SpyTag + SpyCatcher‐GRFT complex formed with 44 µg mL−1 (magenta), 171 µg mL−1 (blue), 228 µg mL−1 (green), and 298 µg mL−1 (red) of SpyCatcher‐GRFT. The control groups were CsgA‐SpyTag fibers only and CsgA‐SpyTag fibers incubated with E. coli BL21 (DE3) cell lysate. Initial TCID50 value for SARS‐CoV‐2 was TCID50 = 107.25 mL−1. Data are presented as mean ± SD, n = 3, p‐values are calculated using one‐way ANOVA with Sidak's correction, **p < 0.01, ***p < 0.001.

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) Imaging of Captured SARS‐CoV‐2 on CsgA‐SpyTag + SpyCatcher‐GRFT Complex

The examination of SARS‐CoV‐2 virus since the beginning of the pandemic has revealed many regarding affected tissues, mechanism of replication, and morphology. The morphology and size of SARS‐CoV‐2 virus particles have been used for imaging the attachment and internalization mechanisms.[ ] Recent publications have investigated SARS‐CoV‐2 via Scanning and Transmission Electron Microscopy (SEM and TEM) methods and shown that the virus particles have a diameter around 70–100 nm.[ , , ] Moreover, CsgA biofilm fibers form interwoven mesh structures,[ ] with individual fibers having approximate widths of 4–6 nm.[ , ] In more recent studies the width of single fibers was also reported to be as wide as 25–35 nm.[ ] Regarding the morphological features of the SARS‐CoV‐2 virus particles and the complex structure mainly composed of CsgA‐SpyTag biofilm fibers, they are comparable to each other under SEM and TEM.

Subsequent to infectivity analysis of the supernatant after CsgA‐SpyTag + SpyCatcher‐GRFT complex was incubated with SARS‐CoV‐2, we have investigated the pelleted fiber complex in order to verify the virus capture. As it was concluded from the infectivity analysis, the CsgA‐SpyTag + SpyCatcher‐GRFT fiber complex pellet interacted and captured the SARS‐CoV‐2 virus particles via GRFT and left significantly low infectious viruses in the supernatant. SEM and TEM images of the complex pellet corroborate the SARS‐CoV‐2 capture by the complex structure. The samples were prepared from the complex structure containing 171 µg mL−1 CsgA‐GRFT due to the lowest infectivity results after incubation (Figure 3b). The SEM and TEM images were taken for pellets obtained after CsgA‐SpyTag + SpyCatcher‐GRFT complex and CsgA‐SpyTag fibers were separately incubated with SARS‐CoV‐2 virus. The CsgA‐SpyTag bare fibers were used to be able to verify the effect of GRFT in the complex structure. The SEM images of CsgA‐SpyTag + SpyCatcher‐GRFT complex incubated with SARS‐CoV‐2 indicate attachment of the virus particles to the fiber structure of the complex (Figure  ) as indicated by arrows. On the contrary, for the control group of only CsgA‐SpyTag fibers, the virus particles were observed to be distant from the CsgA‐SpyTag fibers (Figure 4d,e). This confirms the effect of GRFT on the capture of SARS‐CoV‐2 virus. Furthermore, the TEM images provided a closer look at the samples. TEM image of the pelleted complex structure after incubation period with SARS‐CoV‐2 demonstrated a single virus particle captured on the mesh structure of CsgA‐SpyTag + SpyCatcher‐GRFT fiber complex, which has a diameter of almost 70 nm (Figure 4c). The TEM image for CsgA‐SpyTag fibers only provided the mesh structure of CsgA‐SpyTag fibers without any SARS‐CoV‐2 virus captured (Figure 4f).

Figure 4

SEM and TEM images of pellets obtained after CsgA‐SpyTag + SpyCatcher‐GRFT complex and bare CsgA‐SpyTag fibers were incubated with SARS‐CoV‐2. The CsgA‐SpyTag − SpyCatcher‐GRFT complex was selected according to the results of the infectivity assay. The complex having 171 µg mL−1 CsgA‐GRFT was chosen to be investigated due to its least infective properties. a) SEM image of CsgA‐SpyTag + SpyCatcher‐GRFT pellet with SARS‐CoV‐2 virus particles (diameter is around 70 nm) attached to the fibers. Scale bar represents 500 nm. b) Zoomed in SEM image for the framed zone (black dashed line). Scale bar represents 300 nm. c) TEM image for CsgA‐SpyTag – SpyCatcher‐GRFT pellet with SARS‐CoV‐2 virus particles. Scale bar represents 50 nm. d) SEM image of bare CsgA‐SpyTag fiber pellet incubated with SARS‐CoV‐2 virus particles (diameter is 50–70 nm). Scale bar represents 500 nm. e) Zoomed in SEM image for the framed zone (black dashed line). The SARS‐CoV‐2 virus particles were not in close vicinity of bare CsgA‐SpyTag fibers as they are for CsgA‐SpyTag + SpyCatcher‐GRFT complex fibers. Scale bar represents 300 nm. f) TEM image for bare CsgA‐SpyTag pellet. No SARS‐CoV‐2 virus particles were observed. Scale bar represents 50 nm.

Lateral Flow Assay for SARS‐CoV‐2 Precipitation via CsgA‐SpyTag + SpyCatcher‐GRFT Complex

The detection of SARS‐CoV‐2 infection is achieved by RT‐PCR as it is denoted as the golden standard due to its high accuracy and sensitivity.[ ] However, in circumstances where resource limitation is an issue for accessibility of RT‐PCR, Lateral Flow Assays (LFAs) have been developed for rapid SARS‐CoV‐2 infection detection.[ ] In such limited resource scenarios, the detection of SARS‐CoV‐2 infections and the attempts to improve the detection are of crucial importance. Therefore, CsgA‐SpyTag + SpyCtatcher‐GRFT complex system was assessed in terms of any improvement it can provide in LFA test results. Our purpose was to observe the capability of the complex system to accelerate the precipitation of SARS‐CoV‐2 through the bottom of sample tubes along with itself so that it would create a difference in the results. The LFA tests were courtesy of Synbiotik Biotechnology.

Subsequent to analysis of virus infectivity and capture via CsgA‐SpyTag + SpyCatcher‐GRFT complex, we investigated the precipitation ability of the complex structure. Five clinical samples with known Ct values (Table S1, Supporting Information) were incubated separately with CsgA‐SpyTag + SpyCatcher‐GRFT complex structure. At the end of the incubation period, the supernatant and complex pellet were separated from each other and both were diluted with LFA buffer prior to loading to cassettes (Figure  ). The capture of SARS‐CoV‐2 via CsgA‐SpyTag + SpyCatcher‐GRFT complex and the precipitation through the complex pellet structure provides an observable difference in the LFA detectable SARS‐CoV‐2 amount. The cassettes labeled as S1, S2, S3, S4, and S5 represent supernatants of clinical samples separated from the complex pellet after incubation and centrifugation step. The LFA results indicated the supernatants still contained a detectable amount of SARS‐CoV‐2 virus particles. On the other hand, the cassettes labeled as P1, P2, P3, P4, and P5 are complex pellets separated from supernatants latter to incubation and were tested with LFA cassettes for the captured SARS‐CoV‐2. The LFA cassettes run for supernatants and complex pellets resulted in different intensities of test lines (Figure 5b,c, red arrows). According to the band intensities analyzed with ImageJ software, the complex pellet band intensities are almost twofolds greater than that of supernatant bands for cassettes 1, 2, 3, and 4 (Figure S2, Supporting Information). For cassette 5, the clinical sample has a relatively low Ct value indicating higher viral load, which may have been the reason to have relatively closer band intensities for supernatant and complex pellet since all the virus particles may not be captured by the complex. For cassette C (the control group, 6 as denoted in Figure S2, Supporting Information), the supernatant and complex pellet band intensities are very close. The control group (C) was composed of bare CsgA‐SpyTag fibers and incubated with one of the clinical samples. The results obtained from control did not show as much difference between supernatant and the bare CsgA‐SpyTag fiber pellet in terms of detectable SARS‐CoV‐2 as those taken after incubation with CsgA‐SpyTag + SpyCatcher‐GRFT. As a result, the calculated band intensity differences between supernatant and complex pellet helped to imply that CsgA‐SpyTag + SpyCatcher‐GRFT complex has the ability to be used as a precipitation mechanism for SARS‐CoV‐2 in aqueous media.

Figure 5

LFA experiment for SARS‐CoV‐2 precipitation via CsgA‐SpyTag + SpyCatcher‐GRFT complex. a) The clinical samples obtained by nasopharyngeal swabs were mixed and incubated with CsgA‐SpyTag + SpyCatcher‐GRFT complex. Following incubation of the complex and SARS‐CoV‐2 virus from clinical swabs, supernatant and complex pellet were separated and analyzed using LFA. The figure is created with BioRender. b) S1, S2, S3, S4, and S5 represent supernatants taken after five clinical samples were separately incubated with CsgA‐SpyTag + SpyCatcher‐GRFT complex. c) P1, P2, P3, P4, and P5 represent complex pellets that were separated from the supernatants of five clinical samples. The test lines (red arrows) for supernatant and complex pellets were observed to be different from each other in terms of intensity indicating that the CsgA‐SpyTag + SpyCatcher‐GRFT complex is capable to precipitate the SARS‐CoV‐2 viruses from the supernatant to some extent. The red asterisk (*) represents bare CsgA‐SpyTag fibers incubated with SARS‐CoV‐2 as control (C).

Following the validation that our complex system is able to capture and concentrate SARS‐CoV‐2 virus particles, we aimed to analyze the sensitivity of CsgA‐SpyTag + SpyCatcher‐GRFT complex for lower concentration of viruses. Therefore, we used 6 positive (tested by local health institute through RT‐PCR) clinical samples with varying Ct values (Table S2, Supporting Information). The CsgA‐SpyTag + SpyCatcher‐GRFT structure itself was also tested on LFA cassettes (Figure  ).

Figure 6

LFA experiment for assessing the sensitivity of CsgA‐SpyTag + SpyCatcher‐GRFT complex against lower concentration viruses in clinical samples. a) The obtained 6 clinical samples were mixed with LFA extraction buffer provided by the Synbiotik Biotechnology along with the LFA cassettes. Each of the clinical samples were mixed with CsgA‐SpyTag + SpyCatcher‐GRFT complex and incubated for 30 min. Following the incubation period, the complex pellet was separated from the supernatant by centrifuging. b) The supernatant was mixed with LFA buffer (1:7) and loaded to LFA cassettes. c) The pelleted complex structures were resuspended with LFA buffer and loaded to LFA cassettes. d) CsgA‐SpyTag + SpyCatcher‐GRFT complex was also tested alone as a control that it does not contribute to the positive results.

Initially, all the obtained 6 clinical samples were run on LFA cassettes (Figure 6a). The samples 1D, 1F, 1H with Ct values 32.59, 27.39, 30.02, respectively did not resulted as positive in the LFA cassettes due to their relatively high Ct values. Subsequent to the incubation of the clinical samples with the complex structure, the complex pellet and the supernatant were run on LFA cassettes, with the aim of creating a difference due to the captured SARS‐CoV‐2 virus particles in the pellet. The LFA cassettes run for the supernatants latter to incubation displayed a similar trend with that of the samples on their own (Figure 6b). Remarkably, the complex pellets that were centrifuged after the incubation period, showed drastic differences in terms of detecting clinical samples with low concentration SARS‐CoV‐2. The LFA cassettes run from the complex pellets of each sample improved the test band intensities. For the samples 1D, 1F, and 1H, the rapid antigen tests resulted positive upon loading their complex pellets to the LFA cassettes due to concentrating the virus particles with the help of CsgA‐SpyTag + SpyCatcher‐GRFT complex (Figure 6c). As a result, it can clearly be interpreted that the complex structure CsgA‐SpyTag + SpyCatcher‐GRFT is sensitive against clinical samples with high Ct values around 27 and 32 as for the 1D, 1F, and 1H samples. The complex structure poses great opportunities for detecting and diagnosing the clinical samples with low virus concentrations for test mechanisms less sensitive than RT‐PCR such as rapid antigen tests.

Conclusion

We here developed a genetically engineered biomaterial that uses E. coli major curli protein CsgA as the scaffold and embraces functional groups which are fused to the scaffold through SpyTag–SpyCatcher interactions. The developed complex structure can be modified with varying functional groups each of which serve varying purposes. In the studied complex structure, our aim was to capture and isolate the SARS‐CoV‐2 virus particles from aqueous media through the antiviral lectin protein GRFT as the functional group. We have shown that via CsgA‐SpyTag + SpyCatcher‐GRFT complex system the capture of the SARS‐CoV‐2 virus is achieved along with the reduction in the infectivity of the virus due to the antiviral lectin protein, GRFT. We have further analyzed the complex structure for its capabilities in concentrating the SARS‐CoV‐2 virus particles in clinical samples for testing in lateral flow assay antigen tests. We have observed detectable changes regarding the test line intensities in LFA tests for the pellet harboring the complex structure with the captured virus particles and the supernatant, which relatively contains fewer virus particles. In addition, different approaches to use GRFT for capturing and isolating SARS‐CoV‐2 viruses may be present, such as the integration of GRFT to solid matrices through conjugation mechanisms. However, the coupling procedures of proteins to beads or solid matrices require chemical conjugation between the protein and the solid matrix of interest. When compared to our system, the chemical coupling of GRFT to beads or other solid matrices may pose problems regarding the successful attachment of GRFT to the beads. On the other hand, in our system we rely on the protein–peptide interaction happening between SpyCatcher and SpyTag domains, which result in an irreversible isopeptide bond formation. The coupling of SpyCatcher‐GRFT fusion protein to the CsgA‐SpyTag biofilm protein happens as a result of this isopeptide bond and compared to chemical conjugation options, we believe our strategy is more powerful. We have concluded that the developed CsgA‐SpyTag + SpyCatcher‐GRFT complex system as a genetically engineered biomaterial is capable of interacting, capturing, and concentrating the SARS‐CoV‐2 virus particles from aqueous media culminating in a significant reduction in the total infectivity and an observable difference in the LFA detection tests. Last but not least, the complex system is highly adaptable for numerous purposes due to its modularity. Therefore, the developed complex system can be modified to serve purposes for instance environmental detection, pollution prevention, and more.

Experimental Section

Plasmids for Constructing Fiber Complex Subunits

The CsgA‐SpyTag construct was designed by fusing SpyTag peptide tag to the C‐terminus of CsgA with a GS linker in between. On the other hand, for the SpyCatcher‐GRFT construct, SpyCatcher protein was fused to the N‐terminus of GRFT protein, having a GS linker in between. The genes encoding CsgA‐SpyTag and SpyCatcher‐GRFT fusion proteins were cloned into pZa vector under tetO promoter and pET22b (+) vector under T7 promoter, respectively. pZa CsgA‐SpyTag and pET22b (+) SpyCatcher‐GRFT plasmids were then transformed into ΔcsgA strain derived from E. coli (MG 1655) and BL21 (DE3) strain, respectively.

Preparation of SpyCatcher‐GRFT Fusion Protein

E. coli BL21 (DE3) cells accommodating pET22b SpyCatcher‐GRFT plasmid were inoculated in fresh LB medium with ampicillin (30 µg mL−1) and incubated overnight at 37 °C, 200 rpm. After overnight incubation, cells were diluted 1:100 into fresh LB medium supplied with ampicillin (30 µg mL−1). The cells were grown until optical density (OD600) reached 0.4 and induced with IPTG (1 mm). The cells were incubated at 16 °C, 200 rpm overnight. The cells were pelleted via centrifugation at 8000×g for 5 min, and the supernatant was removed.

To lyse the cells, the pellet was resuspended in 1X Phosphate Buffer Saline (PBS, pH 7.0) at a dilution rate of 1:10. Three rounds of freeze‐thaw cycle were applied using liquid nitrogen, and sonication was conducted for 5 min with 10 s ON and 10 s OFF cycles with an amplitude of 35%. The sonicated cells were centrifuged at 13000×g for 45 min, and the supernatant was filtered using a 0.20 µm pore size cellulose acetate syringe filter (VWR).

Preparation of CsgA‐SpyTag Fibers

ΔcsgA cells harboring pZa tetO CsgA‐SpyTag plasmid were inoculated into fresh lysogeny broth (LB) medium supplied with 30 µg mL−1 ampicillin and incubated overnight at 37 °C, 200 rpm. Following overnight incubation, the cells were diluted 1:100 into fresh LB medium with 30 µg mL−1 ampicillin and grown until OD600 reached 0.4. Once the OD600 condition was achieved, the cells were pelleted at 8000×g for 5 min, and the supernatant was removed. The cell pellet was resuspended in fresh 1X M63 minimal medium (pH 7.0) supplied with MgSO4 (1 mm), glycerol (0.2% v/v), casein hydrolysate (0.1% v/v), thiamine (1 µg mL−1), and ampicillin (30 µg mL−1). The cells were incubated at 30 °C incubator in stationary condition and induced with aTc (250 µg mL−1) every two days.

At the end of the induction period, the CsgA‐SpyTag fiber was filtered as described earlier.[ ] The cell culture was incubated with 0.8 m Guanidine‐Hydrochloride (Gdn‐HCl) for 2 h at 4 °C and filtered through a 47 mm polycarbonate filter membrane having 10 µm pores (EMD Millipore). The residue on the filter membrane was incubated with 5 mL of Gdn‐HCl (8 m) for 5 min and vacuum filtered. The filter membrane was rinsed with 5 mL dH2O and vacuum filtered. The residue on the filter membrane was incubated with 5 mL of SDS (5% m/v) for 5 min and vacuum filtered. The membrane was again rinsed with 5 mL of dH2O and vacuum filtered. The biofilm fiber residues on the filter membrane were collected with a spatula (Figure S3a,b, Supporting Information).

SpyTag − SpyCatcher complex formation with CsgA‐SpyTag and SpyCatcher‐GRFT fusion proteins was conducted as described earlier[ ] latter to filtration and lysis processes. CsgA‐SpyTag fibers were arranged to have a final concentration of 1 mg mL−1, whereas SpyCatcher‐GRFT fusion protein was determined to be 4 different concentrations (44, 171, 228, 298 µg mL−1) when forming fiber complexes (Figure S4, Supporting Information). The complex formation was carried out in Phosphate Buffer (50 mm) (1 m KH2PO4, 1 m K2HPO4, pH 7.2). CsgA‐SpyTag was incubated with SpyCatcher‐GRFT in Phosphate Buffer (50 mm) at 4 °C overnight. The CsgA‐SpyTag + SpyCatcher‐GRFT fiber complex samples were collected at the end of the incubation period by centrifuging at 14 000×g for 5 min. The supernatant was removed from the CsgA‐SpyTag + SpyCatcher‐GRFT fiber complex. The fiber complexes were washed 3 times with dH2O to remove non‐specific SpyCatcher‐GRFT fusion protein from the environment.

SARS‐CoV‐2 Infectivity Assay after Incubation with Fiber Complex

Virus infectivity assay was conducted with Tissue Culture Infective Dose 50% (TCID50) method, and all infectivity assays with SARS‐CoV‐2 were performed in Biosafety Level 3 plus (BSL‐3 +) laboratory. To elucidate the binding capacity of CsgA‐SpyTag + SpyCatcher‐GRFT fiber complex with SARS‐CoV‐2 virus particles in a suspension of CsgA‐SpyTag + SpyCatcher‐GRFT fiber complexes with varying SpyCatcher‐GRFT as described earlier. The fiber complexes were mixed and incubated with equal volumes of SARS‐CoV‐2 (TCID50 = 107.25 mL−1) virus for 30 min. The mixtures were centrifuged at 14 000×g for 10 min at the end of the incubation period. Supernatants were then transferred into sterile tubes and titrated in vitro for the infectivity assay as previously conducted.[ ]

Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM)

The filtered CsgA‐SpyTag fiber pellets were resuspended with dH2O to have a final concentration of 1 mg mL−1. Formvar‐Carbon coated TEM grids (200 mesh nickel grids, Electron Microscopy Sciences) were initially incubated with fiber samples for 5 min. Then, TEM grids were washed with dH2O and Selective Binding Buffer (SBB, 1X PBS, 300 mm NaCl, 80 mm imidazole, 0.2% (v/v) Tween‐20) 5 min, respectively. The grids were incubated with 10 nm 5 nm Ni‐NTA gold nanoparticles in the dark for 90 min at room temperature. Following incubation, TEM grids were washed for 5 min with SBB, PBS, and dH2O for 5, 2, and 2 times, respectively. TEM grids were incubated with uranyl acetate (UA) (2%) for 25 s and left for drying at room temperature. The grids were then visualized with 200 kV TEM (FEI Tecnai).

SARS‐CoV‐2 capture by CsgA‐SpyTag + SpyCatcher‐GRFT complexes was also visualized by TEM. The samples of fiber complexes with captured SARS‐CoV‐2 virus particles were incubated with Formvar‐Carbon TEM grids and left for drying and, therefore inactivation overnight in the Biosafety cabinet. The grids were stained with UA (2%) for 25 s and left for drying at room temperature in biosafety cabinet. The grids were then visualized with 200 kV TEM (FEI Tecnai).

The samples of SARS‐CoV‐2 mixed and incubated with CsgA‐SpyTag + SpyCatcher‐GRFT complex were dropped on silica wafers and left for drying overnight in Biosafety cabinet. Air drying the droplets led the inactivation of SARS‐CoV‐2 in the samples without disturbing its physical state. Later, the silica wafers were attached to stubs with carbon tape. The samples were then coated with Au/Pd of 10 nm thickness using Precision Etching and Coating System (PECS) since the samples were not conductive in their natural states. Finally, the samples were visualized in SEM/FIB (FEI Nova NanoLab 600i SEM/FIB).

Western Blot

SpyCatcher‐GRFT samples were dissolved in 1X SDS loading dye (Laemmli Sample Buffer) and incubated at 95 °C for 5 min prior to loading. CsgA‐SpyTag + SpyCatcher‐GRFT complex samples were incubated in 100% Hexafluoro isopropanol (HFIP) overnight prior to addition of 2X SDS loading dye and incubated at 95 °C for 5 min.

The samples were analyzed using 15% gel. The samples were analyzed by Western Blot by transferring the proteins on the gel to a polyvinylidene difluoride (PVDF) membrane (Thermo Scientific) using an electroblotting system (Bio‐Rad). The PVDF membrane was incubated with 5% blocking buffer (non‐fat dried milk in Tris Buffered Saline, 0.1% Tween‐20, TBS‐T) at 4 °C overnight on a shaker to block non‐specific bindings. PVDF membrane was then incubated with anti‐his mouse primary antibody (Thermo Scientific Pierce) in TBS‐T (1:5000), for 1 h at room temperature on a shaker. The PVDF membrane was washed 3 times with TBS‐T. Then, the membrane was incubated with anti‐mouse horseradish peroxidase (HRP) conjugated secondary antibody in TBS‐T (1:10 000). The membrane was washed again with TBS‐T 3 times. The PVDF membrane was visualized with Vilbert Lourmat imaging system after incubation with an enhanced chemiluminescence (ECL) substrate (Biorad).

Lateral Flow Assay

200 µL of clinical samples were mixed and incubated with CsgA‐SpyTag + SpyCatcher‐GRFT complex structure for 30 min in microcentrifuge tubes. During the incubation period the tubes were inverted several times. At the end of incubation period, the tubes were centrifuged at 14000 × g for 10 min and the supernatant was separated from the CsgA‐SpyTag + SpyCatcher‐GRFT complex pellet. The supernatant was diluted 1:7 with LFA buffer and 70 µL was loaded to the sample port of each cassette. The pelleted CsgA‐SpyTag + SpyCatcher‐GRFT complex was resuspended with LFA buffer and 70 µL was loaded to the sample port of the cassettes. The results were obtained within 5 mins. All the procedure was conducted in Biosafety Cabinet. LFA cassettes and buffer were courtesy of Synbiotik Biotechnology. All the clinical samples were transferred in BioNAT buffer.

Statistical Analysis

Data expressed as mean ± SD. For analyzing the significance between data sets, one‐way ANOVA testing followed by a Sidak's multiple comparisons test was carried out across groups. In all cases, significance was defined as p ≤ 0.05. Statistical analysis was carried out using GraphPad Prism‐6 Software.

Conflict of Interest

The authors declare no conflict of interest.

Supporting Information

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