Dataset Information

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 D1-D3 protein from unpublished study was used as control.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.

SUBMITTER: Faghri J

PROVIDER: S-EPMC3586881 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Publications

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

Faghri Jamshid J Shahbazzadeh Delavar D Pooshang Bagheri Kamran K Moghim Sharareh S Ghasemian Safaei Hajieh H Nasr Esfahani Bahram B Fazeli Hossein H Yazdani Rahmatolah R Mirmohammad Sadeghi Hamid H

Iranian journal of basic medical sciences 20120301 2

<h4>Objectives</h4>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 vacc ...[more]

PMID: 23493747

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Project description:The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene (TF) encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Staphylococcus simulans Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The TF gene was expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1L8823-MCS-CP3 vector, and in Escherichia coli using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a TF gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 (TF gene) to 0.93 (TFnt gene). Incorporation of the TFnt gene into the pEAQ-HT vector, followed by transient expression in N. benthamiana, led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive Staphylococcus aureus 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against S. aureus 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC50 25 µg/ml) and 60% (IC50 71 µg/ml) growth inhibition against S. aureus 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.

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Dataset Information

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

Objectives

Materials and methods

Results

Conclusion

Publications

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

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