Autotransporter-based surface expression and complementation of split TreA fragments utilized for the detection of antibodies against bovine leukemia virus

https://doi.org/10.1016/j.jim.2021.113084Get rights and content

Abstract

Bovine Leukemia Virus (BLV) is an oncogenic virus which is the etiological agent of a neoplastic disease in infected cattle called enzootic bovine leukemia (EBL). The most common and sensitive diagnostic methods for EBL like enzyme-linked immunosorbent assay (ELISA) is time-consuming and requires manual handling which makes it unsuitable as an on-farm diagnostic test. Hence, there is a need for an alternative test with rapid detection and reduced manual labour. We have previously reported the use of E. coli periplasmic trehalase (TreA) in a split enzyme sensor diagnostic technology to detect immunoglobulins and antigen-specific antibodies. In the current study, a more sensitive detection was attempted by bacterial surface display of split TreA fragment by fusion with the autotransporter AIDA-I. The split TreA fragments fused to antigens require antigen-specific antibodies for complementation and to trigger trehalase activity. This surface complementation strategy was used to detect anti-BLV antibodies in clinical serum by incorporating the antigenic BLV capsid protein in the fusion proteins. To validate this assay, a panel of serum samples obtained from BLV positive and negative cattle were tested in comparison with ELISA results. Evaluation of this panel resulted in positive detection of all true positive samples. We further demonstrated that this assay can be enhanced by pre-adsorption of clinical serum samples using E. coli cells to increase the specificity and help reduce nonspecific binding. In conclusion, the p24 antigen specific BLV assay is a potential tool for simple and rapid diagnosis of BLV infection, which is compatible with both lab-based and a more user friendly on-farm format.

Introduction

Bovine leukemia virus (BLV), a delta retrovirus, is the causative agent of a lymphoproliferative disease in cattle, Enzootic Bovine Leukosis (EBL) (Gillet et al., 2007; Radostits et al., 2007). It is common in North American dairy cattle and accounts for considerable economic losses to the dairy industry (Bartlett et al., 2014; Bartlett et al., 2013; Gillet et al., 2007), due to reduced longevity, decreased milk production and limitations on exports, immune dysfunction, and premature culling (MacLachlan and Dubovi, 2011). The commonly used serological diagnostics that have been developed for BLV are agar gel-immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). Currently, ELISAs to detect antibodies against gp51, an envelope glycoprotein or the major structural core protein p24, are the most common techniques to diagnose BLV infection (De Giuseppe et al., 2004; Stear, 2005). ELISA is a robust and reliable assay for the detection of the presence of BLV in clinical samples due to its sensitivity and specificity (Bai et al., 2018). However, a major limitation of ELISA is the requirement of manual handling and the long processing time (hours) which makes it unsuitable as on-farm diagnostic test. Hence, there is a need for an alternative test with rapid detection and reduced manual labour. We have developed a novel split enzyme-based biosensor platform which is effective in the detection of bovine IgGs, antigen-specific antibodies and protein aggregation (Drikic and De Buck, 2018). The split enzyme sensor uses E. coli glycolytic enzyme trehalase (TreA) which hydrolyzes the disaccharide trehalose, into two glucose molecules. Glucose is detected by a colorimetric enzymatic assay using glucose oxidase, horseradish peroxidase and the substrate o-dianisidine. For the purpose of the biosensor, the trehalase was split into two fragments, the N-terminal fragment, TreAN and the C-terminal fragment, TreAC. These fragments assemble only when fused to interacting protein pairs (antibody with antigen) leading to the activity of the enzyme being restored which hydrolyses trehalose (substrate) into two molecules of glucose (Drikic and De Buck, 2018).

In the current study, we report the expression of the split TreA fragments fused to antigens on the outer membrane (OM) of E. coli cells using the AIDA-I autotransporter system. The expression of proteins in the OM of E. coli reduces the time and expense associated with protein purification (Jose et al., 2012). The autotransporter AIDA-I utilizes the Sec mediated system in bacteria to transport the passenger pre-peptide to the periplasm. The C-terminal translocation unit of AIDA-I tethers to the outer membrane forming a β-barrel pore through which the linker and passenger protein pass unfolded (Jarmander et al., 2012). Gene expression of this autotransporter is driven by the IPTG-inducible lacUV5 promoter and includes two protease cleavage sites for the purpose of surface expression analysis (Shakeel et al., 2020).

We present the successful implementation of bacterial surface expression by displaying the full periplasmic TreA on the surface and validating the functional activity of the full enzyme. Secondly, surface expression and complementation of split TreA fragments fused to streptococcal protein G (pG) was used to detect bovine IgGs. Finally, the surface complementation strategy was applied in the development of a test to detect BLV antibodies in clinical serum.

Section snippets

Reagents and antibodies

4× Na-maleate buffer stock (0.2 M) (pH 6) was made by adding 23.22 g of Maleic Acid (Sigma-Aldrich, Oakville, Canada). and 14.0 g of Sodium hydroxide (Sigma-Aldrich) in 1 l ddH2O.

Monoclonal antibodies (anti-HIS-tag mAb) were purchased from MBL Corporation (Woburn, MA), anti-p24 polyclonal serum was generated by immunizing rabbits with 100 μg/mL of a hybrid antigenic protein, a non-assembling mutant of BLV capsid protein p24 fused to peptide fragments of P12 (KEGHWARDCPTKATG, WARDCPTKATGPPPG,

Expression of full periplasmic TreA on the OM

Expression of the full E. coli periplasmic trehalase, treA, was performed to demonstrate that the full enzyme can be successfully expressed and folded on the surface. The ΔABC strain was used for protein production and functional assays as the wild type BL21 strain can hydrolyze trehalose under high and low osmolarity conditions using the genes TreA, TreB and TreC for the enzymes involved in the trehalose utilizing pathways. The expression of E. coli TreA was confirmed by separating proteins

Discussion

The main goal of the present study was to modify the existing biosensor platform developed in our laboratory by successfully expressing and complementing split TreA fragments on the OM. The surface complementation strategy was then utilized in an assay format to detect BLV antibodies in polyclonal serum and clinical serum samples. We have previously reported the split enzyme complementation of E. coli glycolytic enzyme trehalase (TreA), that hydrolyses trehalose into two glucose molecules,

Conclusions

In conclusion, we demonstrated that the split TreA p24 assay is a valuable tool in the detection of anti-BLV antibodies in clinical serum and can distinguish between sero-positive and sero-negative samples. Further testing and modifications of this assay is clearly required prior to field testing like using pre-assembled freeze-dried reagents and a commercially available handheld glucometer for detection of the output signal, testing blinded and or randomized field samples. Further adaptations

Funding

This work was supported in part by Alberta Agriculture and Forestry, grant # 2016F072R and NSERC CRD, grant # CRDPJ 536202 - 18

Declaration of Competing Interest

None.

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