Caractérisation d'anticorps monoclonaux dirigés contre l'Alpha-Hémolysine de Staphylococcus aureus

In this study, vaccine-based targeting of Hemolysin Alpha (HLA) provides protection against lethal staphylococcal infection in a murine model system, suggesting that a monoclonal antibody-based therapy may likewise prove to be efficacious for prevention and treatment of this infection. We report the generation of four distinct anti-α-hemolysin monoclonal antibodies (MAbs) that antagonize toxin activity in vitro and protecting experimental animals against lethal S. aureus infection.AURE

1H, 13C and 15N backbone resonance assignments of the β-lactamase BlaP from Bacillus licheniformis 749/C and two mutational variants

Class A β-lactamases have been widely used as versatile scaffolds to create hybrid (or chimeric) proteins for a series of applications ranging from basic research to medicine. We have, in particular, used the β-lactamase BlaP from Bacillus licheniformis 749/C (BlaP) as a protein scaffold to create model polyglutamine (polyQ) proteins in order to better understand the mechanism(s) by which an expanded polyQ sequence triggers the formation of amyloid fibrils. The model chimeras were designed by inserting a polyQ sequence of various lengths at two different locations within BlaP (i.e. position 197 or position 216) allowing a detailed comparison of the effects of subtle differences in the environment of the polyQ sequence on its ability to trigger protein aggregation. In order to investigate the effects of the polyQ insertion at both positions on the structure, stability and dynamics of BlaP, a series of NMR experiments including H/D exchange are foreseen. Accordingly, as necessitated by these studies, here we report the NMR assignment of the wild-type BlaP (BlaP-WT) and of the two reference proteins BlaP197Q0 and BlaP216Q0, wherein a dipeptide Pro-Gly has been introduced at position 197 and 216, respectively; this dipeptide originates from the addition of the Sma1 restriction site at the genetic level to allow further polyQ sequence insertion

The ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class a penicillin-binding protein

soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli (14)C(-)labeled lipid 11 precursor. As a DD-peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k(2)/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs

Peptidase activity of b-lactamases

Although b-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of b-lactamases. The kcat/Km values were below 0.1 M-1·s-1, but the enzyme rate enhancement factors were in the range 5000–20000 for the best substrates. Not unexpectedly, the best 'peptidase' was the class C b-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-D-Ala-D-Ala dipeptides than with the diacetyl- and a-acetyl-L-Lys-D-Ala-D-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble DD-peptidases. A comparison between the b-lactamases and DD-peptidases showed that it might be as difficult for a DD-peptidase to open the b-lactam ring as it is for the b-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations

Peptidase Activity of Beta-Lactamases

Although beta-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of beta-lactamases. The kcat/Km values were below 0.1 M(-1). s(-1), but the enzyme rate enhancement factors were in the range 5000-20000 for the best substrates. Not unexpectedly, the best 'peptidase' was the class C beta-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-d-Ala-d-Ala dipeptides than with the diacetyl- and alpha-acetyl-l-Lys-d-Ala-d-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble dd-peptidases. A comparison between the beta-lactamases and dd-peptidases showed that it might be as difficult for a dd-peptidase to open the beta-lactam ring as it is for the beta-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations

A recombinant alpha-hemolysin polypeptide of Staphylococcus aureus, having deletion in the stem domain and heterologous sequences inserted

publication date: 2010-07-22; filing date: 2010-01-1

Specificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly-L-Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D-Ala-D-Ala peptidases. The Gly-L-Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly-L-Xaa peptide or D-Alanine are supplied as acceptors. Utilization of substrates in which the terminal -COO− group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibilty of the transpeptidation reaction

Enzymatic functionalization of a nanobody using protein insertion technology

Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has be- come a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase consti- tutes a suitable technology to functionalize nanobodies and allowsthe creation of versatile tools that can be used in innovative biotechnological assays