Nanobody-Functionalized Cellulose for Capturing SARS-CoV-2

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 253 million people, claiming ∼ 5.1 million lives to date. Although mandatory quarantines, lockdowns, and vaccinations help curb viral transmission, there is a pressing need for cost-effective systems to mitigate the viral spread. Here, we present a generic strategy for capturing SARS-CoV-2 through functionalized cellulose materials. Specifically, we developed a bifunctional fusion protein consisting of a cellulose-binding domain and a nanobody (Nb) targeting the receptor-binding domain of SARS-CoV-2. The immobilization of the fusion proteins on cellulose substrates enhanced the capture efficiency of Nbs against SARS-CoV-2 pseudoviruses of the wildtype and the D614G variant, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose to capture viruses from complex fluids in a continuous fashion. By capturing and containing viruses through the Nb-functionalized cellulose, our work may find utilities in virus sampling and filtration towards paper-based diagnostics, environmental tracking of viral spread and reducing viral load from infected individuals. IMPORTANCE The ongoing efforts to address the COVID-19 pandemic center around the development of diagnostics, preventative measures, and therapeutic strategies. In comparison to existing work, we have provided a complementary strategy to capture SARS-CoV-2 by functionalized cellulose materials towards paper-based diagnostics as well as virus filtration in perishable samples. Specifically, we developed a bifunctional fusion protein consisting of both a cellulose-binding domain and a nanobody specific for the receptor-binding domain of SARS-CoV-2. As a proof-of-concept, the fusion protein-coated cellulose substrates exhibited enhanced capture efficiency against SARS-CoV-2 pseudovirus of both wildtype and the D614G mutant variants, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography for binding viruses from complex biological fluids in a highly continuous and cost-effective manner. Such antigen-specific capture can potentially immobilize viruses of interest for viral detection and removal, which contrasts with the common size- or affinity-based filtration devices that bind a broad range of bacteria, viruses, fungi, and cytokines present in blood ( https://clinicaltrials.gov/ct2/show/NCT04413955 ). Additionally, since our work focuses on capturing and concentrating viruses from surfaces and fluids as a means to improve detection, it can serve as an “add-on” technology to complement existing viral detection methods, many of which have been largely focusing on improving the intrinsic sensitivities.

A rapid simple point-of-care assay for the detection of SARS-CoV-2 neutralizing antibodies

Background Neutralizing antibodies (NAbs) prevent pathogens from infecting host cells. Detection of SARS-CoV-2 NAbs is critical to evaluate herd immunity and monitor vaccine efficacy against SARS-CoV-2, the virus that causes COVID-19. All currently available NAb tests are lab-based and time-intensive. Method We develop a 10 min cellulose pull-down test to detect NAbs against SARS-CoV-2 from human plasma. The test evaluates the ability of antibodies to disrupt ACE2 receptor—RBD complex formation. The simple, portable, and rapid testing process relies on two key technologies: (i) the vertical-flow paper-based assay format and (ii) the rapid interaction of cellulose binding domain to cellulose paper. Results Here we show the construction of a cellulose-based vertical-flow test. The developed test gives above 80% sensitivity and specificity and up to 93% accuracy as compared to two current lab-based methods using COVID-19 convalescent plasma. Conclusions A rapid 10 min cellulose based test has been developed for detection of NAb against SARS-CoV-2. The test demonstrates comparable performance to the lab-based tests and can be used at Point-of-Care. Importantly, the approach used for this test can be easily extended to test RBD variants or to evaluate NAbs against other pathogens.

A T9SS Substrate Involved in Crystalline Cellulose Degradation by Affecting Crucial Cellulose Binding Proteins in Cytophaga hutchinsonii

Cytophaga hutchinsonii is an abundant soil cellulolytic bacterium that uses a unique cellulose degradation mechanism different from those that involve free cellulases or cellulosomes. Though several proteins were identified to be important for cellulose degradation, the mechanism used by C. hutchinsonii to digest crystalline cellulose remains a mystery. In this study, chu_0922 was identified by insertional mutation and gene deletion as an important gene locus indispensable for crystalline cellulose utilization. Deletion of chu_0922 resulted in defect in crystalline cellulose utilization. The Δ 0922 mutant completely lost the ability to grow on crystalline cellulose even with extended incubation, and selectively utilized the amorphous region of cellulose leading to the increased crystallinity. As a protein secreted by the type Ⅸ secretion system (T9SS), CHU_0922 was found to be located on the outer membrane, and the outer membrane localization of CHU_0922 relied on the T9SS. Comparative analysis of the outer membrane proteins revealed that the abundance of several cellulose binding proteins, including CHU_1276, CHU_1277, and CHU_1279, was reduced in the Δ 0922 mutant. Further study showed that CHU_0922 is crucial for the full expression of the gene cluster containing chu_1276 , chu_1277 , chu_1278 , chu_1279 , and chu_1280 ( cel9C ), which is essential for cellulose utilization. Moreover, CHU_0922 is required for the cell surface localization of CHU_3220, a cellulose binding protein that is essential for crystalline cellulose utilization. Our study provides insights into the complex system that C. hutchinsonii uses to degrade crystalline cellulose. IMPORTANCE The widespread aerobic cellulolytic bacterium Cytophaga hutchinsonii , belonging to the phylum Bacteroidetes , utilizes a novel mechanism to degrade crystalline cellulose. No genes encoding proteins specialized in loosening or disruption the crystalline structure of cellulose were identified in the genome of C. hutchinsonii , except for chu_3220 and chu_1557 . The crystalline cellulose degradation mechanism remains enigmatic. This study identified a new gene locus, chu_0922 , encoding a typical T9SS substrate that is essential for crystalline cellulose degradation. Notably, CHU_0922 is crucial for the normal transcription of chu_1276 , chu_1277 , chu_1278 , chu_1279 , and chu_1280 ( cel9C ), which play important roles in the degradation of cellulose. Moreover, CHU_0922 participates in the cell surface localization of CHU_3220. These results demonstrated that CHU_0922 plays a key role in the crystalline cellulose degradation network. Our study will promote the uncovering of the novel cellulose utilization mechanism of C. hutchinsonii.

A Single Mutation in the Carbohydrate-Binding Module Enhances Cellulase Activity in Bacillus Amyloliquefaciens Mutant

From our earlier work, we modified the carbohydrate-binding module (CBM) of Bacillus amyloliquefaciens to increase cellulase activity using cold plasma technology. The cellulase gene (BglC-M) from the mutant was expressed in Escherichia coli BL21(DE3) under the T7 promoter. The hydrolysis activity of the cellulase mutant (BglC-M) was approximately 2.5-fold higher than the control (BglC-W) over a wide range of pH and temperature conditions. The amino acid sequence of the mutant BglC-M contained 471 residues that were almost identical to the control BglC-W. Only a single amino acid, lysine, was replaced by glutamic acid at position 370 (K370E) within the carbohydrate-binding module (CBM). Structure prediction and substrate docking of BglC-M indicated that the single mutation (K370E) might involve cellulose binding of the β-sandwich facilitated by hydrogen bonding. The docking study of cellopentaose with the model structure of BglC-M indicated that the replacement of lysine-370 led to the formation of a hydrogen bond with 436Y, which has a shorter distance (2.6 Å) compared with the control (5.4 Å). As a result, the structure becomes more compact and stable, resulting in increased catalytic efficiency. Finally, the biomass hydrolysis ability of cellulase was investigated on lignocellulosic wastes such as pineapple peel, corncob, and durian peel. The BglC-M enzyme showed a more significant amount of reducing sugar released from all lignocellulosic wastes than the control. This was the first evidence that altering the base composition of the cellulose binding module enhanced the catalytic activity. HIGHLIGHTS Increasing cellulase activity of Bacillus amyloliquefaciens using plasma technology Mutation at cellulose-binding module enhance cellulase hydrolysis activity Greater cellulase activity in the hydrolysis on lignocellulosic wastes GRAPHICAL ABSTRACT

Nanobody-Functionalized Cellulose for Capturing and Containing SARS-CoV-2

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 217 million people, claiming ~ 4.5 million lives to date. Although mandatory quarantines, lockdowns, and vaccinations help curb viral transmission, safe and effective preventative measures remain urgently needed. Here, we present a generic strategy for containing SARS-CoV-2 by cellulose materials. Specifically, we developed a bifunctional fusion protein consisting of a cellulose-binding domain and a nanobody (Nb) targeting the receptor-binding domain of SARS-CoV-2. The immobilization of the fusion proteins on cellulose substrates enhanced the capture efficiency of Nbs against SARS-CoV-2 pseudoviruses of the wildtype and the D614G variant, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose for neutralizing virus from contaminated fluids in a continuous and cost-effective fashion. Taken together, our work leverages low-cost cellulose materials and recently developed Nbs to provide a complementary approach to addressing the pandemic.

Nanobody-Functionalized Cellulose for Capturing and Containing SARS-CoV-2

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 196 million people, claiming ~ 4.2 million lives to date. Although mandatory quarantines, lockdowns, and vaccinations help curb viral transmission, safe and effective preventative measures remain urgently needed. Here, we present a generic strategy for containing SARS-CoV-2 by cellulose materials. Specifically, we developed a bifunctional fusion protein consisting of a cellulose-binding domain and a nanobody (Nb) targeting the receptor-binding domain of SARS-CoV-2. The immobilization of the fusion proteins on cellulose substrates enhanced the capture efficiency of Nbs against SARS-CoV-2 pseudoviruses of both the wildtype and the D614G variant, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose for neutralizing virus from contaminated fluids in a continuous and cost-effective fashion. Taken together, our work leverages low-cost cellulose materials and recently developed Nbs to provide a complementary approach to addressing the pandemic.

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation

Lignocellulosic biomass from plants has been used as a biofuel source and the potent acidic endoglucanase GtCel12A has been isolated from Gloeophyllum trabeum, a filamentous fungus. In this study, we established a plant-based platform for the production of active GtCel12A fused to family 3 cellulose-binding module (CBM3). We used the signal sequence of binding immunoglobulin protein (BiP) and the endoplasmic reticulum (ER) retention signal for the accumulation of the produced GtCel12A in the ER. To achieve enhanced enzyme expression, we incorporated the M-domain of the human receptor-type tyrosine-protein phosphatase C into the construct. In addition, to enable the removal of N-terminal domains that are not necessary after protein expression, we further incorporated the cleavage site of Brachypodium distachyon small ubiquitin-like modifier. The GtCel12A-CBM3 fusion protein produced in the leaves of Nicotiana benthamiana exhibited not only high solubility but also efficient endoglucanase activity on the carboxymethyl cellulose substrate as determined by 3,5-dinitrosalicylic acid assay. The endoglucanase activity of GtCel12A-CBM3 was maintained even when immobilized on microcrystalline cellulose beads. Taken together, these results indicate that GtCel12A endoglucanase produced in plants might be used to provide monomeric sugars from lignocellulosic biomass for bioethanol production.