FACING Staphylococcus aureus INFECTIONS THROUGH CHARACTERIZATION OF RECOMBINANT ANTIBODIES AGAINST THE COLLAGEN ADHESIN CNA BY EPITOPE MAPPING (Affrontare le infezioni da Staphylococcus aureus attraverso la caratterizzazione di anticorpi ricombinanti contro l’adesina per il collagene CNA mediante epitope mapping)

Staphylococcus aureus is one of the major human pathogens despite being a commensal of nasal and skin flora. This bacterium is notorious for its ability to become resistant to antibiotics. Acquisition of resistance to antibiotics by pathogens makes infection treatment complicated, increases health care costs, morbidity and mortality. Infections caused by antibiotic-resistant strains like methicillin-resistant Staphyloccocus aureus (MRSA) are indeed well documented in clinical setting. The spreading of pathogenic strains also in normal community is of great medical interest, as it limits treatment options for affected patients. Since antibiotic resistance is increasing with a much faster rate than the introduction of new drugs for infection treatment, alternative or complementary therapies are necessary to be developed. Adhesion to host cells is the first and critical step to establish infection. This step happens thanks to expression of a repertoire of surface proteins, named adhesins, that recognize components of extracellular matrix such as fibrinogen, fibronectin and collagen. The use of anti-adhesive monoclonal antibodies, able to interfere with the binding of matrix components may be an interesting approach to face antibiotic resistance problem. To this aim, in this master thesis work, the characterization of twenty human monoclonal antibodies generated by phage display technique was pursued. These antibodies were selected against the staphylococcal collagen adhesin CNA (in particular against the fragment displaying the minimum collagen binding activity of CNA, CNA-(151-318)), since it has been shown to be a relevant virulence factor in many different animal models of arthritis, keratitis, endocarditis, mastitis and osteomyelitis. Preliminary studies demonstrated that nine antibodies were only CNA-(151-318) binders, seven bound CNA-(151-318) and CNA-(31-344), three antibodies bound not only to CNA but also to at least one other bacterial collagen adhesins. Moreover, one antibody showed both inhibiting and displacing activity on CNA-collagen binding. To better define the properties of these monoclonal antibodies, we investigated on the nature of the epitope by ELISA and SPR analysis in order to map the epitopes exploiting chimeric proteins. ACE, the enterococcal collagen adhesin structurally related to CNA, was used as inert template for constructing chimeric proteins for epitope mapping where regions of CNA-(151-318) replaced the corresponding sequences of ACE-(152-318). Comparing data from both approaches we could discover that two antibodies map to the N-terminal quarter, eight mAbs map to the central region and eight map to the C-terminal end of CNA-(151-318).