The Bacterial Surface Expression of SARS Viral Epitope using Salmonella typhi Cytolysin A

Abstract

The cytolysin A (ClyA) is a 34 kDa pore-forming cytotoxic protein and expressed by some enteric bacteria including Salmonella typhi. This toxin is transported on the bacterial surface and secreted without posttranslational modification. Using the surface display of ClyA, the expression vectors for 193-aa immunogenic antigen of spike protein (termed S1E) from severe acute respiratory syndrome coronavirus (SARS-CoV) were constructed. The vectors carried a gene encoding S. typhi ClyA conjugated to S1E at the C terminus (termed ClyA-S1E) and asd gene in pGEM-T and pBR322, named pGApLCS1E and pBApLCS1E, respectively. An asd-mutated E. coli transformed with these vectors could grow without diaminopimelic acid (DAP), indicating that they were stably maintained in such mutants. ClyA-S1E recombinant proteins from these vectors were expressed on the surface of the attenuated S. typhimurium deficient of global virulence gene regulator, ppGpp. However, they did not show the hemolytic activity on the blood agar plate and cytotoxicity against HeLa cells. To examine whether bacteria expressing ClyA-S1E induced the immune response against S1E, S. typhimurium deficient of ppGpp and Asd was transformed with these vectors and orally immunized in mice. In the western blotting against GST-conjugated S1E using the immunized mouse sera, it was shown that the significant band was detected in the mouse serum by the bacteria transformed with pGApLCS1E but not with pBApLCS1E. It indicates that the immune response producing antibody was dependent on the expression level of ClyA-S1E. Therefore, ClyA delivery system can be used for SARS vaccine development.

Inhibition of Tumor Growth and Metastasis by a Combination of Escherichia coli–mediated Cytolytic Therapy and Radiotherapy

“…The slides were incubated with primary antibody, rabbit anti-ClyA (1:80, distributed by Y.H. 48 ) overnight at 4 °C, followed by incubation with antirabbit immunoglobulin G labeled with biotin (Sigma, St Louis, MO), at room temperature for 80 minutes. Subsequently, streptavidin-horseradish peroxidase (Dako, Glostrup, Denmark) detection system was applied.…”

Cytolysin A-mediated protein exportation efficiency and its role in enhancing the fitness of live recombinant Salmonella Typhi vaccine strain

“…Although clyA (34 kDa) was designed to fuse to the antigen, the expression of T. gondii antigens were detected in its clyA‐fusion or nonfusion condition as previously reported (Piao et al . 2009; Huang et al . 2016); the cleavage mechanism of clyA fusion protein remains unknown (Roderer and Glockshuber 2017).…”

“…As a limitation of this study, electron microscopy imaging should be included to confirm the presence of OMVs and localization of the antigens. We may link the variation in the clyA-exportation efficiency to the antigen's molecular character, which could alter the conformation of clyA oligomeric assembly (Piao et al 2009). The crystal structure of clyA protein revealed that it has a negatively charged interior, which is cation-selective (Ludwig et al 1999;Willems et al 2017).…”

Genetically Engineered Salmonella typhimurium as an Imageable Therapeutic Probe for Cancer

“…The expression plasmid pBAD-RLuc8 has been previously described (11). The clyA gene was amplified with 5′-AGTCCATGGTTATGACCGGAATATTTGC-3′ (forward primer) and 5′-GATGTTTAAACTCAGACGTCAGGAACCTC-3′ (reverse primer) using the S. typhi genomic DNA as a template (12). Amplified DNA was cut by NcoI and PmeI and used to directly replace RLuc8 at the same site in pBAD-RLuc8.…”