Dataset Information

The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis

ABSTRACT: Protein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 μg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12934-021-01701-4.

SUBMITTER: Ghaedmohammadi S

PROVIDER: S-EPMC8596801 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundGiven the role of spA as a pivotal virulence factor decisive for Staphylococcus aureus ability to escape from innate and adaptive immune responses, one can consider it as an object subject to adaptive evolution and that variations in spA may uncover pathogenicity variations.ResultsThe population genetic structure was deduced from the extracellular domains of SpA gene sequence (domains A-E and the X-region) and compared to the MLST-analysis of 41 genetically diverse methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains. Incongruence between tree topologies was noticeable and in the inferred spA tree most MSSA isolates were clustered in a distinct group. Conversely, the distribution of strains according to their spA-type was not always congruent with the tree inferred from the complete spA gene foreseeing that spA is a mosaic gene composed of different segments exhibiting different evolutionary histories. Evidences of a network-like organization were identified through several conflicting phylogenetic signals and indeed several intragenic recombination events (within subdomains of the gene) were detected within and between CC's of MRSA strains. The alignment of SpA sequences enabled the clustering of several isoforms as a result of non-randomly distributed amino acid variations, located in two clusters of polymorphic sites in domains D to B and Xr (a). Nevertheless, evidences of cluster specific structural arrangements were detected reflecting alterations on specific residues with potential impact on S. aureus pathogenicity.ConclusionsThe detection of positive selection operating on spA combined with frequent non-synonymous mutations, domain duplication and frequent intragenic recombination events represent important mechanisms acting in the evolutionary adaptive mechanism promoting spA genetic plasticity. These findings argue that crucial allelic forms correlated with pathogenicity can be identified by sequences analysis enabling the design of more robust schemes.

Dataset Information

The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis

Supplementary Information

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets