Simple Affinity-Based Method for Concentrating Viruses from Wastewater Using Engineered Curli Fibers

Abstract

Wastewater surveillance is a proven method for tracking community spread and prevalence of some infectious viral diseases. A primary concentration step is often used to enrich viral particles from wastewater prior to subsequent viral quantification and/or sequencing. Here, we present a simple procedure for concentrating viruses from wastewater using bacterial biofilm protein nanofibers known as curli fibers. Through simple genetic engineering, we produced curli fibers functionalized with single-domain antibodies (also known as nanobodies) specific for the coat protein of the model virus bacteriophage MS2. Using these modified fibers in a simple spin-down protocol, we demonstrated efficient concentration of MS2 in both phosphate-buffered saline (PBS) and in the wastewater matrix. Additionally, we produced nanobody-functionalized curli fibers capable of binding the spike protein of SARS-CoV-2, showing the versatility of the system. Our concentration protocol is simple to implement, can be performed quickly under ambient conditions, and requires only components produced through bacterial culture. We believe this technology represents an attractive alternative to existing concentration methods and warrants further research and optimization for field-relevant applications.

Graphical Abstract

INTRODUCTION

Wastewater-based epidemiology (WBE) is a powerful approach for infectious disease surveillance and has gained significant interest in light of the COVID-19 pandemic.¹⁻⁷ Viruses and viral nucleic acids shed in fecal matter by infected individuals provide a signal for the presence and spread of infections in a given population.^8,9 Analysis of population pooled wastewater can therefore aid public health decision making and serve as an early warning system for disease outbreaks emerging at the community level.^5,6,9−14 WBE also has the potential to circumvent the biases of clinical data, which can be influenced by the availability of diagnostic tests, physical or financial access to healthcare facilities among the local population, testing policies and regimens, and lag times between infection and clinical testing.¹⁵ Although the recent pandemic has invigorated WBE research, the benefits of wastewater surveillance have been well recognized since it was used in the 1990s to help eradicate poliovirus.¹⁶

Virus levels in wastewater are often measured through quantification of viral nucleic acids via the quantitative polymerase chain reaction (qPCR).^7,8 An important step in most protocols is the concentration of the virus into a smaller sample volume from which nucleic acids are extracted.¹⁷ This is done to improve detection sensitivity, which is often hampered by the relatively low number of viral particles in the sample and other sample characteristics such as high turbidity and high concentrations of organic matter and heavy metals.^8,17

Numerous methods have been developed for concentrating viruses from environmental water samples. One of the most common methods is charge-based adsorption to a filter membrane or filter media followed by elution or direct nucleic acid extraction.¹⁸⁻²⁰ These approaches are frequently used in conjunction with a secondary concentration step due to the large elution volumes necessary for efficient viral recovery.²⁰⁻²² Size-based separation techniques such as centrifugal ultrafiltration, hollow fiber ultrafiltration, tangential flow filtration, and ultracentrifugation are also popular.²³⁻²⁶ Another class of concentration methods, which rely on entrapment of viral particles in chemical precipitates, includes PEG-NaCl precipitation, aluminum hydroxide flocculation, and skim milk flocculation.²⁶⁻²⁹ However, many of the above approaches have associated downsides, including filter clogging, long processing times, variable recovery rates, and/or high costs.^30,31 Poor or unpredictable performance in some of these techniques may be attributed, in part, to their nonspecific nature, which prevents differentiation between specific viruses and other species that may be present in the highly heterogeneous wastewater matrix.

Protein nanofibers offer a possible alternative approach to virus concentration through components manufactured from simple bacterial culture. Curli fibers are functional amyloid nanofibers which form a major structural component of Escherichia coli biofilms.³² They are insoluble and resistant to degradation by proteases and detergents.³³ Because curli fibers can be self-assembled from a single protein, CsgA, and have a dedicated secretion system, they are a convenient scaffold for material synthesis and customization through genetic engineering. As a result, curli fibers have attracted great interest as a platform for the development of functional materials.³⁴⁻⁴¹ In particular, their high surface area resulting from nanofibrous morphology has motivated development for sequestration applications.^42,43 Recently, biofilms containing engineered curli fibers were used to capture influenza virus from river water.⁴⁴

Here, we report a simple protocol using engineered curli fibers to concentrate viruses from wastewater, representing an affinity-based alternative to existing viral concentration methods.

METHODS

Strains and Plasmids.

E. coli strain PQN4 (MC4100, Cm^R, ΔcsgBACEFG) was used for curli expression. E. coli used for propagating and quantifying MS2 was American Type Culture Collection (ATCC) 15597. Bacteriophage MS2 was ATCC 15597-B1. Plasmids used in the study are listed in the Supporting Information (Table S1).

Propagation of MS2.

E. coli strain ATCC 15597 was grown in 50 mL of ATCC medium 271 in a shaking incubator at 37 °C and 225 rpm until midexponential phase (OD₆₀₀ = 0.4−0.6). The culture was then inoculated with an individual MS2 plaque and incubated overnight to undergo cell lysis. The lysed culture was transferred to a 50 mL Falcon tube and centrifuged at 500g and 4 °C for 20 min. The supernatant was then filtered through a 0.22 μm filter to remove any remaining E. coli. The filtered supernatant stock was diluted in PBS and stored at 4 °C.

Plaque Assay for Quantifying Viable MS2.

Bottom agar plates were prepared using ATCC medium 271 with 1% (w/v) agar. Top agar was prepared using ATCC medium 271 with 0.3% (w/v) agar, thoroughly boiled in a microwave, and cooled to 52 °C in a water bath. A MS2-containing sample was serially diluted in PBS. Here, 100 μL of an overnight culture of host strain ATCC 15597, 100 μL of MS2-containing sample, and 3 mL of melted top agar were added to the bottom agar plate and swirled to mix. Top agar was solidified at room temperature for 20 min. Plates were incubated overnight at 37 °C to allow for E. coli growth and plaque formation. MS2 concentration was computed based on the number of plaques and the dilution factor.

Curli Fiber Expression.

E. coli strain PQN4 was inoculated from single colonies into 2 mL of lysogeny broth (LB) media supplemented with an antibiotic and incubated in a shaking incubator at 37 °C and 225 rpm (rpm) overnight. The following day starter cultures were diluted 1:100 into fresh LB supplemented with an antibiotic and 0.001% (w/w) arabinose to induce curli fiber expression. Cultures were then grown for 20 h in a shaking incubator at 37 °C and 225 rpm prior to spin-down assays.

Collection and Processing of Wastewater Samples.

Wastewater samples were obtained from a manhole in Somerville, Massachusetts, using a GLS compact composite sampler programmed to collect a 120 mL of wastewater sample every 30 min for 24 h. Occasionally, if the pump failed on site, a grab sample was taken. Wastewater was pasteurized by incubation in a 60 °C water bath for 30 min prior to spiking with MS2.

MS2 Spin-down Protocol.

Here, 20 mL of E. coli culture was transferred to a 50 mL Falcon tube. E. coli cells were spun down at 4000g for 10 min, resuspended in 20 mL PBS, spun down again, and resuspended in 1 mL of PBS. Then, 20 mL of MS2 solution was added at a final concentration of 1.6 × 10⁶ or 1.6 × 10³ PFU/mL. This MS2 solution was prepared by spiking from refrigerated stock into PBS or pasteurized wastewater. For follow-up experiments, the pasteurized wastewater was prefiltered using a 0.22 μm filter or prediluted 1:1 in PBS before spiking. The cell−virus mixture was incubated on a rotating platform for gentle agitation at room temperature for 90 min. The mixture was spun down again, and the supernatant was discarded. The pellet was used for RNA extraction.

Extraction and Purification of MS2 RNA Following Spin Down.

Following MS2 spin down, the E. coli cell pellet was resuspended with a pipet in 1 mL of Trizol reagent. For the positive control representing 100% recovery, MS2 was spiked directly into 1 mL of Trizol to reflect the total amount of MS2 which had been added during the spin-down assay. Trizol samples were incubated at room temperature for 10 min, then transferred to a 2 mL Eppendorf tube. Here, 200 μL chloroform was added, and the tube was vigorously shaken by hand for 15 s. The mixture was incubated at room temperature for 2 min. The sample was then spun down at 12,000g at 4 °C for 15 min. The aqueous supernatant was removed by pipet and mixed with an equal volume of 70% ethanol. The sample was then transferred to the spin cartridge from a PureLink RNA Mini Kit (Life Technologies). Kit instructions for purification from Trizol-extracted samples were followed. The sample was eluted in RNase-free water and stored at −80 °C prior to quantification by qPCR.

qPCR to Quantify MS2 RNA.

qPCR reactions were prepared by combining a TaqMan Fast Virus 1-Step Master Mix (4X) (Applied Biosystems), forward and reverse primers, a 5′6-FAM/3′ TAMRA probe, and nuclease-free water, such that forward and reverse primers reached final concentration 400 nM, and the probe reached final concentration 200 nM. The TaqMan Fast Virus 1-Step Master Mix was diluted 1:3. This mixture was then divided into 20 μL aliquots in a 96-well reaction plate, and 2 μL of the RNA sample was added for each reaction. Here, 22 μL reactions were prepared in triplicate for each sample. A five-point standard curve using MS2 RNA of known concentration (Roche 10165948001) was included on each plate. Wells for standards and negative controls containing nuclease-free water were also run in triplicate. qPCR was performed using a QuantStudio 3 Real-Time PCR System (Applied Biosystems) thermocycler. Cycling conditions were as follows: reverse transcription for 30 min at 48 °C, denaturation and activation for 10 min at 95 °C, and two-step cycling 15 s at 95 °C and 60 s at 60 °C for 45 cycles. Primer and probe sequences targeting the RNA replicase ß chain (GenBank Accession No. NC_001417) were taken from the literature.⁴⁵ Forward and reverse primer sequences were GCTCTGAGAGCGGCTCTATTG and CGTTATAGCGGACCGCGT. The probe sequence was 5′6-FAM/CCGAGACCAATGTGCGCCGTG/3′TAMRA. Minimum information for publication of the quantitative real-time PCR experiment (MIQE) documentation is detailed in the Supporting Information (Tables S2 and S3).

Spike Protein Spin Down and Whole-Cell ELISA.

1 mL of E. coli culture was transferred to a 2 mL Eppendorf tube. E. coli cells were spun down at 4000g for 5 min, resuspended in 1 mL PBS, spun down again, and resuspended in 1 mL of PBS with 5% BSA. The mixture was incubated end-over-end at room temperature for 5 min. Then, 1 μL of recombinant SARS-CoV-2 spike protein subunit S1 with hFc and His tags (Abclonal RP01259) was added to reach a final concentration of 0.2 μg/mL. The mixture was incubated end-over-end at room temperature for 90 min. The E. coli cells were spun down and washed twice in 1 mL of PBS. The washed cell pellet was then resuspended in 700 μL solution of HRP-conjugated monoclonal antibody from rat raised against a recombinant human IgG Fc fusion protein (BioLegend 410603). The antibody solution was prepared by diluting purchased stock 1:5000 in Tris-buffered saline with 0.1% Tween-20 (w/v) (TBST). The mixture was incubated end-over-end at room temperature for 90 min. The cells were then spun down and washed twice in 1 mL of TBST. The washed pellet was resuspended in 120 μL of Ultra-TMB-ELISA Substrate Solution (ThermoScientific 34028) and incubated at room temperature for 3 min before the addition of 60 μL of 6 M sulfuric acid to stop the HRP reaction. Finally, the cells and curli fibers were spun down at 18,000g for 2 min, and the supernatant was transferred to a 96-well plate to measure the absorbance at 450 nm using a SpectraMax M5Microplate Reader.

Direct Extraction of SARS-CoV-2 RNA from Wastewater.

For direct extraction, wastewater was first pasteurized by incubation in a 60 °C water bath for 30 min. As a process control, 1.3 μL of bovine coronavirus (bCov) (Merck, single dose 2 mL vial) was spiked per 10 mL of pasteurized wastewater. The bCov spike was prepared by suspending the modified live virus in 2 mL of PBS. Here, 1 mL of wastewater was then combined with 3 mL of Trizol-LS reagent. Samples were then mixed in equal volume with 100% ethanol (final volume = 8 mL) and processed for RNA purification. Sample were filtered using vacuum filtration on the EZ-Vac Vacuum Manifold (Zymo Research) to pass liquid through filter columns provided in the Monarch RNA Cleanup kit (New England Biolabs). After the entire 8 mL was filtered, 1.8 mL of the wash buffer from the Monarch kit was applied to sample filters columns. Filter columns were centrifuged at 16,000g for 1 min to remove any excess liquid, and RNA was eluted into 110 μL of nuclease-free water.

Attempted Concentration of SARS-CoV-2 from Raw Wastewater Using Engineered Curli Fibers.

Here, 30 mL of E. coli culture was transferred to a 50 mL Falcon tube. E. coli cells were spun down at 4000g for 10 min, resuspended in 30 mL PBS, spun down again, and resuspended in 15 mL of PBS. The washed 15 mL of E. coli cell suspension in PBS was mixed with 15 mL of wastewater and incubated on a rotating platform for gentle agitation at room temperature for 90 min. For these samples, the wastewater was not pasteurized in order to prevent denaturation of the spike protein but was spiked with bCov as described above. Following the 90 min incubation, the mixture was spun down, and the supernatant was discarded. RNA was extracted from the pellet in the same manner as in the MS2 experiments. Following the centrifugation of the Trizol−chloroform mixture, the aqueous supernatant was mixed in equal volume with 100% ethanol and processed for RNA purification using the same process described above for direct extraction samples.

qPCR to Quantify SARS-CoV-2 RNA.

qPCR assays were performed using the QuantStudio 3 Real-Time PCR System. A premade primer/probe mixture targeting the SARS-CoV-2 RNA N1 gene (Integrated DNA Technologies, 10006713) was used for quantification. Here, 20 μL qPCR reactions were prepared in triplicate with 10 μL of Luna Universal Probe One-Step Reaction Mix (2X) (New England Biolabs), 1 μL of Luna WarmStart RT Enzyme Mix (20X), 2 μL of SARS-CoV-2 Primer and Probe Mix, and 7 μL of extracted RNA. The final concentrations of primers and probe were 500 and 125 nM, respectively. Here, 96-well plates for qPCR assays were prepared using the epMotion M5073 Liquid Handling System (Eppendorf). A five-point standard curve using SARS-CoV-2 N2 positive control plasmid (linearized) (IDT, 10006625) was included on each plate for quantification of SARS-CoV-2 RNA. Wells for standards and negative controls containing nuclease-free water were also run in triplicate. Cycling conditions were as follows: 1 cycle of 55 °C for 15 min and 95 °C for 1 min and 45 cycles of 95 °C for 15 s and 55 °C for 30 s. Forward and reverse primer sequences were GACCCCAAAATCAGCGAAAT and TCTGGTTACTGCCAGTTGAATCTG. The probe sequence was 5′FAM/ACCCCGCATTACGTTTGGTGGACC/3′-BHQ1. Minimum information for publication of quantitative real-time PCR experiment (MIQE) documentation is detailed in the Supporting Information (Tables S2 and S3).

Statistical Analysis.

All values and error bars reflect the mean ±1 standard error (s.e.) of three independent experiments (n = 3). All P-values were generated from two-sided Student t-tests for comparing two means (n = 3), which were computed using R statistical software. P-values < 0.05 were used to determine significance.

RESULTS AND DISCUSSION

Efficient Concentration of MS2 Using Nanobody-Functionalized Curli Fibers.

To produce curli fibers for viral concentration, we genetically fused CsgA to a single-domain antibody (also known as a nanobody) with high affinity for the coat protein of the bacteriophage MS2 (NbMS2).⁴⁶ MS2 is commonly used as a surrogate for enteric viruses in environmental fate and transport as well as water treatment studies.⁴⁷ MS2 has several properties that make it a useful surrogate for human pathogenic viruses: (1) It can be cultivated and enumerated using quick and inexpensive methods. (2) It is not pathogenic to humans and therefore obviates the need for cumbersome safety measures. (3) Its morphology resembles several human-infective RNA viruses, such as poliovirus and norovirus. For curli fiber expression, we used an E. coli strain PQN4 lacking the csg genes responsible for curli fiber synthesis but containing a plasmid with a synthetic, arabinose-inducible csgBACEFG operon. This allowed us to easily modify the composition of the curli fibers through genetic engineering of the csgA sequence on the plasmid. We note that PQN4 is derived from MC4100 and lacks the genes necessary for production of the F pilus, which serves as the MS2 receptor.⁴⁸ We used a simple spin-down protocol for viral concentration; curli-expressing cells were first coincubated with virus particles, before centrifugation to spin down virus particles captured in the curli-cell network.

We first verified that NbMS2-functionalized curli fibers could be used to sequester viable MS2 particles. Following the spin-down protocol, we observed a significantly greater reduction in the concentration of viable MS2 in the supernatant when the E. coli expressed NbMS2-functionalized curli fibers, compared to negative controls where curli fiber expression was not induced or where the fibers contained CsgA fused to an off-target nanobody (NbGFP) (Figure S1).⁴⁹

Next, we tested whether our concentration protocol could be used to efficiently recover MS2 RNA. Curli-expressing cells from a 20 mL culture were washed in phosphate buffered saline (PBS) and resuspended in a 20 mL solution of MS2 in PBS. The mixture was incubated for 90 min on a rotating platform at room temperature, then centrifuged at 4000g to harvest the E. coli and captured MS2. The MS2-containing pellet was resuspended in 1 mL of Trizol reagent for RNA extraction, followed by RNA purification using a silica column-based kit. Finally, MS2 RNA was quantified using qPCR and compared to a positive control in which the MS2 was spiked directly into Trizol. When using NbMS2-functionalized curli fibers, we observed average recovery efficiencies of 110% and 119% of MS2 RNA at the two MS2 concentrations tested, 1.6 × 10⁶ and 1.6 × 10³ PFU/mL, respectively (Figure 1). In contrast, when using NbGFP-functionalized fibers, we observed average recovery efficiencies of 4% and 0%.

Figure 1.

Efficient concentration and recovery of MS2 RNA. Following the spin-down protocol, MS2 RNA was recovered from the cell pellet by resuspension in Trizol reagent and silica column-based purification. Recovery efficiency was measured by performing qPCR and comparing to a positive control in which MS2 was spiked directly into Trizol from a concentrated stock. The assay was performed in both PBS and pasteurized wastewater (WW). The spiked MS2 concentration was also varied: high and low MS2 indicate 1.6 × 10⁶ and 1.6 × 10³ PFU/mL, respectively. At the low concentration, predilution of WW in PBS significantly improved the recovery efficiency compared to undiluted WW (two-sided Student t-test for two means, P-value = 0.00069). *** P-value < 0.001. Values and error bars reflect the mean ± standard error (s.e.) of at least three independent experiments (3 ≤ n ≤ 7).

During the co-incubation with MS2 in these experiments, E. coli were present at the same cell density as the overnight culture from which they were harvested, i.e., between 2 × 10⁹ and 2.5 × 10⁹ CFU/mL. Therefore, at the higher concentration of MS2, the ratio of bacteria to virus was roughly 1000:1. To test whether recovery efficiency was reduced at a lower ratio, we performed spin-down and recovery experiments using E. coli cell suspensions diluted 10- to 1000-fold from the overnight culture cell density (Figure S2). We found that a 10-fold dilution of the E. coli cell suspension resulted in a concomitant 10-fold decrease in recovery of MS2 RNA, showing that the 1000:1 ratio of bacteria to virus was necessary to efficiently concentrate MS2 using our protocol.

Next, we performed similar experiments with MS2 spiked into wastewater instead of PBS. For these experiments, the wastewater was pasteurized prior to spiking with MS2 as a biosafety precaution due to the possible presence of SARS-CoV-2. The average recovery efficiencies using the NbMS2 fibers decreased to 50% and 7% for the high and low MS2 concentrations, respectively. We hypothesized that pretreatment of the wastewater matrix might improve recovery efficiency. To test this, we measured the recovery efficiency in wastewater samples which were prediluted 1:1 in PBS or prefiltered with a 0.22 μm filter. We found that predilution of wastewater increased recovery efficiency to 120% and 78% at the high and low MS2 concentrations, respectively. In contrast, prefiltration did not significantly improve recovery efficiency at the low concentration. We hypothesized that the curli fibers may be coconcentrating PCR inhibitors present in the wastewater matrix, leading to an apparent increase in recovery efficiency in the diluted samples. However, we found no strong evidence of PCR inhibition in RNA extract samples from either the 100% or 50% wastewater experiments (Figure S3).

We note that the error bars illustrate a high degree of variability in the MS2 recovery data. This variability is likely due, in part, to pipetting errors during the preparation of positive standards for multiple rounds of qPCR and inconsistent yields during the RNA extraction and purification procedure. We also note that several of the NbMS2 samples exhibited recovery efficiencies well over 100%. Again, this could be due to variability inherent to performing multiple rounds of qPCR and RNA extraction. Another possibility is that the presence of the NbMS2 nanobodies enhanced the efficiency of the RNA extraction compared to incubation of the virus in Trizol alone. Nonetheless, these results demonstrate that curli fibers displaying nanobodies can efficiently and specifically concentrate viruses spiked into wastewater samples at environmentally relevant concentrations.⁸

Engineered Curli Fibers Targeting the SARS-CoV-2 Spike Protein.

SARS-CoV-2 RNA was consistently detectable in local municipal wastewater samples at the time of the study, suggesting the presence of SARS-CoV-2 particles as a possible target for our system.⁵⁰ To produce curli fibers targeting SARS-CoV-2, we generated 10 different curli variants in which CsgA was fused to a protein with putative binding affinity for the spike protein of SARS-CoV-2. Eight of these constructs encoded CsgA fused to a nanobody (nb112, NbSARS, D4, H4, Ty1, Sb23, MR3, W25), while the remaining two encoded CsgA fused to sequences derived from the spike protein receptor, ACE2 (Spikeplug and 22–57).⁵¹⁻⁵⁹

To validate the spike protein-binding activity of these engineered curli fibers, we performed a spin-down protocol to capture purified spike protein (S1 subunit) which had been spiked into PBS and used a whole-cell ELISA assay to probe for the presence of captured spike protein in the cell pellet. We found that several of the engineered curli fiber variants showed greater spike protein-binding activity compared to four negative controls (Figure 2). The two fusions with ACE2-derived sequences showed no significant binding activity. The five nanobodies fusions which exhibited the greatest binding activity were nb112, NbSARS, Ty1, Sb23, and MR3. We decided to use these five variants to concentrate SARS-CoV-2 from raw wastewater.

Figure 2.

Spin down of purified, recombinant SARS-CoV-2 spike protein from PBS using engineered curli fibers. A live-cell ELISA assay was developed to probe for the presence of spike protein in the curli-cell pellet following the spin-down protocol. The four bars on the left correspond to various negative controls, and the remaining 10 bars correspond to samples where the curli fibers were engineered to display protein or peptide domains with putative binding affinity for the SARS-CoV-2 spike protein. Higher absorbance readings correspond to increased immobilization of the antibody used to detect the presence of the recombinant spike protein in the cell pellet. Values and error bars reflect the mean ± s.e. of three independent experiments (n = 3).

To concentrate SARS-CoV-2 from wastewater, curli-expressing cells from a 30 mL culture were washed and resuspended in 15 mL of PBS. Here, 15 mL of raw, unpasteurized wastewater was then added to the cell suspension for a final volume of 30 mL. Viral concentration, RNA extraction, and RNA purification were performed similarly to the MS2 experiments. SARS-CoV-2 RNA was then quantified using qPCR. To evaluate our concentration protocol, we compared the amount of SARS-CoV-2 RNA recovered from the curli-cell pellet to the amount of RNA directly extracted from 1 mL of pasteurized wastewater. We found that the concentration protocol was inefficient, with none of the spike protein-targeting nanobody fusions achieving recovery efficiency greater than 17% (Figure S4). Moreover, none of these samples significantly outperformed the NbMS2 negative control. Notably, the ability to detect SARS-CoV-2 RNA following direct extraction from wastewater (without concentration) in these experiments may only be possible because of its abnormally high levels during the pandemic (Figure S5).

CONCLUSIONS

In this work, we developed a novel method for viral concentration using engineered biofilm protein nanofibers. Viral sequestration was mediated by nanobody domains fused to the major curli subunit CsgA that targeted antigens present on the virus surface. We validated our approach by demonstrating efficient recovery of the surrogate virus MS2 from both PBS and wastewater prediluted 1:1 in PBS. The improved concentration efficiency after dilution of wastewater in PBS might be attributed to dilution of inhibitory substances such as detergents. Our protocol is simple to implement, can be performed quickly under ambient conditions, and requires only components produced from bacterial culture, making it an attractive alternative to existing concentration technologies.

We also produced nanobody-functionalized curli fibers capable of binding the SARS-CoV-2 spike protein. However, attempts to concentrate SARS-CoV-2 from wastewater were inefficient. Recent studies have shown that SARS-CoV-2 quickly loses viability in wastewater, while SARS-CoV-2 RNA has greater persistence.^60,61 Another study found that SARS-CoV-2 RNA in wastewater is mostly present in a form accessible to the intercalating dye propidium monoazide.⁶² These findings suggest that a portion of SARS-CoV-2 RNA in wastewater is not contained in an intact viral envelope, which could help explain why the spike protein-mediated concentration was inefficient. Notably, the 4S concentration method, which includes a NaCl-based viral lysis step, was recently developed expressly to co-concentrate RNA from both intact and lysed virus.⁶³ Curli fibers targeting other antigens, such as the nucleocapsid protein (N) or the viral RNA itself, could exhibit increased recovery of RNA from mixtures containing inactivated virus. Additionally, the ability to specifically concentrate intact, infectious SARS-CoV-2 (or other viruses) could be an advantage in applications such as microbial risk assessment. Future studies could evaluate the specificity of our approach for concentrating infectious virus compared to partially degraded virus or naked viral RNA.

The use of protein nanofibers that are easily modified through genetic engineering offers many possibilities for further improvement of this system. Multifunctional curli fiber networks could be engineered to bind multiple targets, either through production of multifunctional fiber networks or through mixing of different fiber variants. Curli-cell networks could be engineered to include covalent cross-linkages to yield hyper-aggregated networks, enabling easier processing of larger volumes without the need for a centrifuge.⁶⁴ The use of light-switchable nanobodies could allow for a catch-and-release system, which would support the recovery of captured virus without direct resuspension in a harsh Trizol buffer.⁶⁵ Additionally, the presence of living E. coli could allow for simultaneous biosensing applications.⁶⁶

One limitation of this study is the use of the bacteriophage MS2 as a surrogate for human pathogenic viruses. Although MS2 is often used as a proxy for enteric viruses, it is less appropriate for approximating the behavior of enveloped viruses such as SARS-CoV-2.²⁹ Moreover, the use of surrogate viruses, in general, can bias estimations of recovery efficiency.⁶⁷ Future studies should evaluate the effectiveness of curli fiber-based viral concentration using relevant human viruses in addition to both enveloped and nonenveloped surrogates.

Overall, given the simplicity of the concentration protocol, the versatility of engineered curli fibers, and the potential sustainability of microbial manufacturing, we believe this technology warrants further research and development for field-relevant applications in surveillance of wastewater and other environmental water matrices.