Two Dimensional Structural Analysis and Expression of a New Staphylococcus aureus Adhesin Based Fusion Protein

Abstract

Objectives: Staphylococcus aureus is a foremost source of numerous nosocomial and community acquired infections. Antibiotic therapy for vancomycin resistant S. aureus (VRSA) can not promise the eradication of infections. Since adhesion is the major route of infections, adhesin based vaccine could suppress S. aureus infections. Fibronectin binding protein A (FnBPA) and clumping factor A (ClfA) are major responsible adhesions involved in S. aureus infections, so they could be candidate vaccine molecules against an extensive range of infections. This project intended to express a new fusion protein construct and analysis of biological activity regarding binding activity.

Materials and methods: pfnbA- ClfA construct was transformed to Escherichia coli BL21 (DE3). Transformant E. coli were grown in LB broth and induced with IPTG and cellular extracts were separated on SDS-PAGE. RT-PCR was performed to verify expression. Binding activity of fusion protein was studied using human gingival fibroblast (HGF) cell line. D1-D3 protein from unpublished study was used as control.

Results: The expected fusion protein fragment showed by SDS-PAGE. RT-PCR verified the existence of mRNA relating to expressed fusion protein. Binding activity of S. aureus decreased after treatment of HGF cells with fusion protein.

Conclusion: In total, binding activity of fusion protein was approximately two fold lesser than D1-D3 protein. It is supposed that the fusion protein could not be attached to its ligand easily and would be more accessible to antigen presenting cells and consequently protective antibodies will be produced. This project is pending for in vivo infection study in animal model.

Keywords: Adhesion; Clumping factor A; Fibronectin binding protein A; Fusion protein; Staphylococcus aureus; Two dimensional structure.

Figure1

Prediction of 2-Dimensional structure of the FnBPA

Figure 2

Prediction of 2-Dimensional structure of the ClfA

Figure 3

2-D structure of D1-D3

Figure 4

2-D structure of terminal segment of ClfA binding domain

Figure 5

2-D structure of recombinant D1-D2-D3-ClfA

Figure 6

SDS-PAGE of crude extracts relating to induced and uninduced cells for fusion protein.

Figure 7

SDS-PAGE of crude extracts relating to induced and uninduced cells for D1-D3

Figure 8

RT-PCR for mRNA related to fusion protein

Figure 9

RT-PCR for mRNA related to D1-D3

Figure 10

Average quantity of attached bacteria /cell in fusion protein group

Figure 11

Average quantity of attached bacteria /cell in D1-D3 group